**Flow Cytometer FACS Calibur, FACS Scan, and FACS Vantage flow sorter**

**Standard Operating Procedures**

FACS Facility

Building 977 -- 106

December 15, 2008

Please Note: These procedures will be updated as needed; with the
current version always posted in the FACS facility, on the
instrumentation. Users need to be ensure that they always use the
current, posted version.

Prepared By: Damir Sudar, Life Sciences Division

> Heidi Feiler, Life Sciences Division

**Table of Contents**

1.  Research Description

    1.  Work Overview

    2.  Flow cytometer and Sorter (FACS) Analysis Summary

    3.  How to Use the BSL2 FACS Facility

2.  Responsibilities

3.  Determination of Hazards and Controls

    1.  Hazards

> Biohazardous Materials
>
> Lasers
>
> Pressure

2.  Controls

> Administrative
>
> Training
>
> Work Practices
>
> Engineering
>
> Biosafety Cabinet
>
> FAC HEPA Exhaust System
>
> Personal Protective Equipment

4.  FACS Calibur and FACSscan Operating Procedure

    1.  System Check

    2.  Running a Sample

5.  FAC Vantage Operating Procedures

    1.  System Check

    2.  Running a Sample

6.  FACS Vantage Preventive Maintenance Procedures

7\. Appendix

> A: FACS Users Log (Excel Spread Sheet)
>
> B: FSE Tools/Supplies Required

**1.0 Research Description**

See the appropriate section of users BUA

**1.1 Work Overview**

Cell flow instruments and the technical support and expertise will be
available to the LBNL and external research community. These instruments
include a Becton-Dickinson (BD) FACS Vantage flow cytometer sorter, a BD
FACS Calibur flow cytometer and a FACSscan. These instruments will sort
or isolate subpopulations of cells from a variety of sources including
cells with specific reporters, stem cells, or primary tissues (blood or
other human or mouse tissues or tumors).

All researchers wishing to have samples processed on these instruments
will transport their FACS-ready samples to the FACS facility in 977-106
following EH&S guidelines for containment and safety. The samples will
not be stored or incubated on site, but immediately processed on the
cytometer/sorter by a well-trained technician, Sarah Elmes (here on an
MLA from the UCSF Laboratory for Cell Analysis).

To maintain a record for EH&S, we require that each user fill out a form
delineating what biological material is being analyzed and to disclose
any potential biohazards. This request form (Appendix A) has been
developed in collaboration with EH&S as a record of material exposure,
and will be kept on file. Requests that entail unknown biohazards are
referred to EH&S for review. No radioactive material is allowed in the
facility. Once FACS users have gone through extensive, certified FACS
training, they may be able to process their own samples.

2.  **Flow cytometer and Sorter (FACS) Analysis Summary**

The Becton-Dickinson (BD) FACS Vantage SE flow cytometer sorter, a BD
FACS Calibur flow cytometer, and a BD FACSscan flow cytometer will
sort/isolate or measure subpopulations of cells from a variety of
sources including cells with specific reporters, stem cells, or primary
tissues (blood or other human or mouse tissues or tumors). Only RG 1 and
2 materials will be processed.

The FACS systems are located in a BSL2 lab in 977-106. Users must ensure
proper protective clothing is worn at all times. While in 977-106,
closed toe shoes and safety glasses are required at all times. When
working with the sorters, or handling or sorting all biological
specimens: closed toe shoes, safety glasses, lab coat, and appropriate
gloves are required at all times.

All researchers wishing to use the FACS facility must ensure that they
have taken all required EH&S training and that the FACS Standard
Operating Procedure document, method of sample transport to the FACs
facility, and trained users have been documented in their PIs BUA,
before scheduling an experiment on the Oracle calendar. When scheduling,
it is required that users give details of the samples that will be
sorted under the "details" tab, listing any potential hazards.

All researchers wishing to have samples processed on these instruments
will transport their FACS-ready samples to the FACS facility in 977-106
following EH&S guidelines for containment and safety as outlined in
their respective PIs BUAs. The samples will not be stored or incubated
on site, but immediately processed on the cytometer/sorter. Researchers,
who have not yet been trained, will schedule processing with the FACS
technician, Sarah Elmes. Researchers, who have passed extensive FACS
training, will be certified by the FACS technician in the Certified User
Log. They will then have their instrument login activated so that they
can process their own samples.

Users are required to fully document and sign-in in the Certified User
Log before each experiment. If this requirement is not met, users will
lose FACS privileges. A computer log will automatically be generated
with each FACS run and checked against the Certified User Log.

To maintain a record for EH&S, before each run each user is required to
sign in to the user log and disclose any potential biohazards. This log
has been developed in collaboration with EH&S as a record of material
exposure, and will be kept on file. If the potential biohazards of a
sample are not well understood, the user is to confer with EH&S prior to
sample processing. No radioactive material is allowed in the facility.

Only trained and authorized users as documented in the Certified User
Log-Book are allowed to operate the FACS systems.

The technician and any trained and authorized users will at all times
follow the FACS Standard Operating Procedures (SOP) for 1) system
operation and 2) running a sample for each of the instruments in the
facility. The SOPs are maintained and available in the FACS facility
with the current master copies posted next to each instrument.
Information in the SOPs may change and all users are expected to
familiarize themselves with any changes.

**1.3 How to Use the BSL2 FACS Facility**

\(1\) The appropriate red \"warning sections\" on protective measures
are at the beginning of each SOP. Users are required to follow these and
all BSL2 safety procedures when they are in the FACS facility.

\(2\) A calendaring step has been added to the summary which requires
calendaring in ORACLE. In the \"details\" section of the entry, the user
describes the sample in enough detail that potential biohazards are
described. 

\(3\) The Certified User Log has been divided into an excel file with 6
tabs.  The first 3 tabs (1 per instrument) is the running list where the
user signs in for a run (columns were added for the user to describe the
sample and potential biohazards, and to put their signature). The last 3
tabs (1 per instrument) is completed by Sarah and is the list of
Authorized/trained users (columns were added for the user and trainer to
put their signatures). 

\(4\) Prior to Training:

> \(a\) Each user checks that they are have completed all EH&S training
> courses to handle RG2 level samples (BSL2 safety procedures are always
> followed in the facility).
>
> \(b\) The user puts a copy of these Standard Operating Procedures in
> their PIs BUA adding a statement of how they will safely transport
> their samples from the PIs laboratory to the FACS facility.
>
> \(c\) If they are trained, they add their name as a \"FACS VANTAGE (or
> FACSscan, or FACS Calibur) user\" on their PIs BUA (next time BUA is
> renewed - until then it is listed in the Certified Log in 106).

\(5\) When a user wants to run:

> \(a\) They calendar their run (describing the potential hazards in
> \"details\")
>
> \(b\) If they will need an Operator for their run, they notify Sarah
> of their run by email (at least a week in advance).
>
> \(c\) The user brings his samples and all needed materials to the
> facility (gloves, tubes, pipettes etc); the user signs in fully on the
> Log sheet.
>
> \(d\) The user verifies that the instrument was left in the correct
> shutdown state; they may want to run a cleaning cycle before they
> start, just to be sure it is ready.
>
> \(e\) The instrument is operated, shut down and cleaned, and the
> samples are processed according to the current, authorized SOP (copy
> on instrument).
>
> \(f\) The user signs out with the time of completion on the Log.
>
> \(g\) The user discards all biohazardous waste in the appropriate
> receptacle (and logs the contents on the sheet affixed to the can),
> discards all other waste and thoroughly cleans up the bench area
> (using a solution of 10% household bleach and then 70% ethanol).
>
> \(h\) Wash hands at the sink before gathering up items and leaving the
> facility.
>
> \(i\) Please let Sarah know if any problems were encountered.

Remember for users to be allowed access to the facility, it is critical
that they practice stringent BSL2 procedures, follow the SOPs, and
thoroughly clean up after themselves.

2.  **Responsibilities**

-   Sarah Elmes will act as the FACS facility manager and be responsible
    for the operation and maintenance of the FACS systems, while Damir
    Sudar and Heidi Feiler provide oversight.

-   FACS users will be responsible for keeping all required EH&S
    training updated and using only the authorized, current FACS SOPs
    posted in the FACS facility. They are also responsible for adding
    the following to their PIs BUA: (1) the FACS Flow Cytometer Sorter
    (FACS) Calibur and Scanner Standard Operating Procedures
    document, (2) documentation of sample preparation and transport to
    the FACS facility, (3) FACS users names and training level.

3.  **Determination of Hazards and Controls**

**3.1 Hazards**

The main hazards associated with use of this equipment involves:
biological materials, lasers and pressure.

Biological Materials:

The projects carried out in the FACS facility (977-106) involve the use
of both Risk Group 1 (RG1) and RG2 biological agents-materials; and will
all be carried out using standard Biosafety Level 2 (BL2) work practice
controls. Personnel handling human tissues, human blood, and human cells
will assume that bloodborne pathogens could be present and will handle
these materials as if they are potentially infectious using "universal
precautions." Personnel handling primary human tissues, cells and
viruses will be very limited and are noted in the body of their PIs BUA.
The transfer of any biological materials to the FACS facility will occur
using BSL2 procedures, including sealed, leak-proof, secondary
containment, gloves, eyeprotection and labcoat. Clean gloves will be
used to open the doors between the labs.

Lasers: The instruments contain embedded lasers to which the class 1
laser protocol applies as approved by LBNL's Laser Safety Officer (LSO).
Under the class 1 embedded laser protocol, only approved and licensed
vendor technicians are allowed open beam alignment and repair
activities. LBNL personnel will NOT perform these activities at any time
but will provide oversight when vendor service personnel performs these
activities. The FACS facility manager will be present at such time.

Pressure: The FACS Vantage utilizes an 80 um nozzle to generate
pressures up to 20-21 psig. Injury may occur from the rupture of high
pressure lines. Additional, materials being sorted can easily be
aerosolized and become a inhalation hazard.

2.  **Controls**

As mentioned previously, aspects of this project are conducted in a
manual fashion in the laboratories listed, and are therefore authorized
by the individual PI's BUAs.

### 

### 3.2.1 Administrative Controls 

### **[Training]{.underline}: Training of all personnel using the FACS will be documented and covered under the BUA issued to their PI. Users are responsible for ensuring that their EH&S training and FACS training is complete and documented in their PIs BUA.** Certified training provided by FACS staff on the FACS instrument of choice will be required before login rights are activated and independent access is allowed. 

[Work Practices]{.underline}:

-   Standard BL2 practices will be used. All human or murine cell lines
    will be handled under BL2 containment.

-   Hand washing will be required.

-   Use of personal protective equipment (gloves, lab coats and safety
    glasses) is required.

-   Surface disinfection (10% solution of household bleach), will be
    performed before and after each run.

-   Agents being removed from these instruments will be either:

> \(1\) handled as biohazardous waste (biohazard waste container and log
> in 977-109),
>
> \(2\) decontaminated by flushing/spraying with a 10% solution of
> household bleach or
>
> \(3\) contained in closed, leak-proof containers for storage,
> centrifugation, or other subsequent processing.

-   The FACs instrumentation/tubing is disinfected by flushing with a
    10% solution of household bleach. Liquid biological waste is mixed
    with household bleach to a 10% concentration and held for at least
    15 min prior to drain disposal.

-   All of the solid biological waste (both RG 1 & 2) is handled as
    biohazardous waste and placed into red biohazard bags, which are
    then taken off site and treated by LBNL's licensed medical waste
    contractor. Bleach and quaternary ammonium compounds are effective
    surface disinfectants against HIV-1 and all cell lines used in this
    project.

-   After work is completed, all work surfaces are sprayed with a 10%
    solution of household bleach and then 70% ethanol.

**3.2.2 Engineer Controls:**

Biosafety Cabinets:

> BL2 containment within a biosafety cabinet or the ACCS unit in 977-116
> will be used for all work involving the growth, treatment,
> transfection and harvesting of human or murine cell lines.

FACS High Efficiency Particulate Air (HEPA) Filtered Exhaust:

> The FACS utilizes a stand-alone HEPA filtering exhaust unit to capture
> any aerosols generated by the FACS machine. To ensure the unit is
> properly operating, the settings on the unit must be checked prior to
> sampling. This unit is also included in the Preventative Maintenance
> (PM) program and must be HEPA tested and certified for use before it
> can be placed back into service.

**3.2.3 Personal Protective Equipment**:

> All personnel working in 977-106 will don the following personal
> protective equipment:

-   Closed toe shoes and safety glasses are required in 977-106. When
    working with the sorters, or handling or sorting all biological
    specimens: closed toe shoes, safety glasses, lab coat, and
    appropriate gloves are required at all times.

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-   Double gloves will be worn at all times when handling unfixed
    primary cells, lentiviral preparations, transfected cells, or the
    combined transfection reagent.

4.  **FACS Calibur and FACSscan Operating Procedures**

    1.  **System Check Protocol**

**[FACS Calibur and FACS Scan SOP]{.underline}**

**[Turn on/off and system check protocol]{.underline}**

**TURN ON:**

-   Main power switch.

-   FACS Flow unit (Calibur only)

-   Mac computer.

To Log-on to the computer:

**Mac:** Username: \<your login\>

Password: \<your password\>

The FACS CellQuest Pro software can be launched at this point.

**START UP DECONTAMINATION**

All work surfaces should be decontamined with an appropriate
disinfectant prior to initiating any work and after the machines have
been place in operation.

**The Stream:**

-   Take the water tube off of the tube holder.

-   Set the system to STAND-BY.

-   Set the dial to RUN. It will drip from the up-take tubing.

To flush the system:

-   Place a cup or plastic holder beneath the syringe. (Next to tube
    holder)

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-   Turn the dial to STAND-BY

-   Hold the syringe plunger in.

-   Rotate the stopcock 180° from pointing to the instrument to pointing
    to yourself.

-   Sheath will pour out from beneath the stopcock. This flushes the
    system of cleaning solutions such as ethanol or bleach and also
    bubbles.

-   Do this for \~30 seconds. The return the stopcock to original
    position. (pointing towards instrument.)

-   To flush the nozzle, put the dial to FILL, press the NOZZLE FLUSH
    button.

-   For more forceful flushing of the nozzle, put the dial to STAND-BY.

-   Rotate the stopcock 90° to point down.

-   The syringe will fill. Keep thumb on the plunger. When full, turn
    the dial to FILL.

-   Push the plunger in.

-   Repeat several times, by turning the dial to STAND-BY to fill the
    syringe and then to FILL\< and pushing on the plunger.

-   With the plunger in the pushed in position, rotate the stopcock back
    to face the instrument.

**Alignment:**

-   Turn dial to STAND-BY.

-   Guide the rainbow beads tube onto the up-take tubing, keeping a drop
    of sheath at the end of the tubing so that no air comes between the
    tubing and the bead solution.

-   Dial to RUN.

-   Use the BD FACS Comp software to calibrate and alignment the system.

**CellQuest Pro SOFTWARE:**

-   If necessary, re-launch the software.

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-   Turn dial to RUN for the rainbow beads.

-   The events will show near the bottom of the screen until you click
    on 'ACQUIRE' . Then they will pop up to where you can see them.

-   Note that on the computer screen in the software, the signal is in a
    'normal position, however on the oscilloscope FSC and/or the SSC
    will be very high.

-   Decrease SSC gain from 4 to 1. Decrease FSC gain from 8 to 1 or 2.

-   In the Break Off /Streams window, under the Break Off tab, turn on
    the Drop Drive.

-   Break Off frequency generally at 46.3.

-   Amplitude at about 16. This may need to be slightly increased
    through the day.

**Calibur or Scan SHUT-DOWN:**

-   Remove sample tube.

-   Install a tube of 10% bleach.

-   Set the system to RUN.

-   Put the system to STAND-BY and remove the bleach.

-   Install a tube of dH2O.

-   Put the pipette contraption (filled with dH2O) under the nozzle so
    that it is slightly submerged. Rest the bottom of the pipette on the
    waste trough. This is so that salt crystals do not dry on the
    nozzle.

-   Turn off the main power switch.

-   Turn off the FACS Flow unit (Calibur only).

**Shut Down Decontamination:**

All work surfaces must be decontaminated with appropriate disinfectant
at the end of each day's run.

**Mac:**

-   Quit the CellQuest Pro software.

-   Shut down the computer.

    1.  **Running a Sample**

**[FACS Calibur and FACS Scan SOP]{.underline}**

**[Running a Sample]{.underline}**

All samples to be run on the BD FACS Calibur or FACSscan must be in a
single cell suspension. Run cells through a 40um filter prior to
arriving at the sorter to achieve this if they are clumping together.

Only 5ml polystyrene round-bottom Falcon tubes can be inserted on the
up-take port. (Cat \# 352054).

-   Uncap sample tube and place cap on an alcohol pad.

-   Make sure the system is set to [Stand-By]{.underline}.

-   Carefully slip the up-take tubing into the sample tube and lift the
    tube up towards the pressure seal.

-   Move the bottom arm beneath the tube. If necessary, adjust the
    height of the base by turning it.

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-   Set the system to [Run]{.underline}.

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-   Press Prime to get the sample moving more quickly through the
    machine.

-   Once events are seen on the plots in the worksheet, you can make
    adjustments to voltages, compensation and threshold values.

-   When all settings are as you want them, click
    '[Record]{.underline}'.

-   Set the dial on the Vantage to '[Standby']{.underline} after the
    file has finished recording.

-   Gates can be set on the populations of interest in the software.

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-   At the end of the run, turn the dial to [Stand-By]{.underline}, cap
    & remove all collection tubes.

-   Proceed with the shut-down procedure.

**5.0** **FAC Vantage Operating Procedures**

**5.1 System Check Protocol**

**[FACS VANTAGE SOP]{.underline}**

**[Turn on/off and system check protocol]{.underline}**

**TURN ON:**

-   Main power switch inside cabinet door.

-   PC computer.

-   House vacuum (typically left on).

-   House Air (typically left on).

-   All work surfaces should be decontamined with an appropriate
    disinfectant prior to initiating any work and after the machines
    have been place in operation.

-   Aerosol Containment Unit with the power button on the front. The
    main power switch at the back on the bottom of the unit should
    always be on.

-   **The magnahelic gauge needle should be around 2. If not, the system
    power level should be changed to bring the indicator to the right
    level. Verify that the magnahelic gauge indicates between the two
    black marks.**

-   Turn on (if needed) 405nm and 633nm lasers power with the control
    switches at the back (note that 488nm laser is powered by main
    system power).

The Digital switch should be **OFF**.

To Log-on to the computers:

**PC:** Ctrl / Alt / Delete

Username: lcacore

Password: lca@2340

Log on to: CC-S171-FACS VANTAGE (This computer)

The FACS DIVA software can be launched at this point.

Turn on Drop Strobe

Turn on Stream Lamp

**The OSCILLOSCOPE:**

Shows pulse display of the parameters

Shows dot plot of the parameters

Shows either single events or the current 100 events

> Sets X & Y parameters on the dot plots. Type both of the parameter
> numbers into the numerical pad, then press this button. The first
> number will be the X parameter. The second number will be the Y.

**The STREAM:**

-   Take the water tube off of the tube holder.

-   Turn on the pressure switch. (Behind the tube holder)

-   If using the 80um nozzle, the sheath pressure should be \~20-21 PSI.

-   The Sample Differential should be \~ 1.2-1.3 for running samples.

-   Turn the dial to FILL then STAND-BY.

-   Adjust the wheel on the door until you see the bottom of the nozzle.

-   Set the dial to RUN. It will drip from the up-take tubing.

To flush the system:

-   Place a cup or plastic holder beneath the syringe. (Next to tube
    holder)

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-   Turn the dial to STAND-BY

-   Hold the syringe plunger in.

-   Rotate the stopcock 180° from pointing to the instrument to pointing
    to yourself.

-   Sheath will pour out from beneath the stopcock. This flushes the
    system of cleaning solutions such as ethanol or bleach and also
    bubbles.

-   Do this for \~30 seconds. The return the stopcock to original
    position. (pointing towards instrument.)

-   To flush the nozzle, put the dial to FILL, press the NOZZLE FLUSH
    button.

-   For more forceful flushing of the nozzle, put the dial to STAND-BY.

-   Rotate the stopcock 90° to point down.

-   The syringe will fill. Keep thumb on the plunger. When full, turn
    the dial to FILL.

-   Push the plunger in.

-   Repeat several times, by turning the dial to STAND-BY to fill the
    syringe and then to FILL\< and pushing on the plunger.

-   With the plunger in the pushed in position, rotate the stopcock back
    to face the instrument.

**ALIGNMENT:**

-   Turn dial to STAND-BY.

-   Guide the rainbow beads tube onto the up-take tubing, keeping a drop
    of sheath at the end of the tubing so that no air comes between the
    tubing and the bead solution.

-   Dial to RUN.

-   To speed the beads through, press the BOOST button.

-   Adjust the Sample Differential to get the beads going trough at
    desired rate, (2-4K / sec)

-   Oscilloscope set to dot plot, SSC v FL1. (Parameters 2 & 3)

-   For 488 laser alignment, you are moving the sort block. Use the X
    dial, the large dial in the front near the X dial, the large dial
    inside the side door, at the top near the left, and the Fluorescence
    Channel wheel.\
    Laser alignment only requires access to the externally accessible
    knobs on the turning towers. **[No open beam alignment is allowed by
    LBNL personnel.]{.underline}** If proper alignment cannot be
    achieved using the alignment knobs, place a service call to BD.

-   Watching to oscilloscope, adjust alignment until the events are
    showing as high and tight as possible in both channels.

-   Align the second and third lasers, one at a time by adjusting the
    specific alignment knobs for each laser. Be sure to change the
    oscilloscope parameters to be reflecting the correct detection
    channel. Again, adjust alignment until the events are showing as
    high & tight as possible.

> **Laser Delay:**

-   Press the Pulse/Sort button next to the oscilloscope to get the
    parameter pusles.

-   Adjust the second laser delay in the Special Set-Up window until the
    faint break-off mark is just to the left of the base of the peak in
    the oscilloscope window.

**DIVA SOFTWARE:**

-   If necessary, re-launch the DIVA software.

-   Turn on the Digital switch.

-   Change oscilloscope to dot plot.

Note: When the DIVA software is not connected, the stream window appears
dark. When it is connected, you can see the illuminated stream window.

Open the following experiment.

\>Browser

\>Admin

\>Tara

\>Laser check exp.

-   Turn dial to RUN for the rainbow beads.

-   Have the oscilloscope set to FSC v SSC.

-   The events will show near the bottom of the screen until you click
    on 'ACQUIRE' . Then they will pop up to where you can see them.

-   Note that on the computer screen in the DIVA software, the signal is
    in a 'normal position, however on the oscilloscope FSC and/or the
    SSC will be very high.

-   Change the monitor input to MAC.

-   Decrease SSC gain from 4 to 1. Decrease FSC gain from 8 to 1 or 2.

-   Leave CELLQUEST running in the back ground until you are finished
    sorting for the day incase further adjustments are necessary.

-   In the BreakOff /Streams window, under the BreakOff tab, turn on the
    Drop Drive.

-   BreakOff frequency generally at 46.3.

-   Amplitude at about 16. This may need to be slightly increased
    through the day.

When the Drop Drive is turned on, noise may appear in the oscilloscope
window. If it looks like FSC noise, adjust the obscuration bar. If it
looks like SSC noise, adjust the two silver dials directly above the
optics block.

Tara has tape on the stream screen near the bottom, with two horizontal
black lines drawn. This is for aligning the first unconnected droplet
between. Usually this will read at about 192 on the screen. Turn the
dial on the sort door to get the first unconnected drop between these
lines.

-   Adjust the amplitude to optimize the last connected droplet and
    minimize the satellite droplets. Generally between 12 -- 20.

    -   Put the charging plates into position, angled out slightly at
        the bottom.

    -   Make sure the stream is going into the center of the waste
        collection trough.

    -   Turn on the voltage on the instrument panel.

    -   Double check the stream is still going into the center of the
        waste collection trough. If not, adjust with the dial.

    -   Turn on the side streams in the BreakOff/Side stream window,
        using the Test Sort button.

    -   The side streams can be dragged in or out using numerical values
        under the Streams Tab.

    -   Check that side & center streams all look tight. It may be
        easier to see if the stream laser is dimmed slightly.

    -   Adjust 2^nd^/3^rd^ & 4^th^ drops if necessary to tighten the
        center stream.

**AREA SCALING:**

-   Set Window extension to '0'

    -   While running the rainbow beads, adjust Area Scaling in an
        appropriate detector for each laser. This means to increase or
        decrease the Area scaling factor until the Area histogram plot
        In DIVA is in the same location as the equivalent Height plot.

    -   Reset window extension to '5'.

**ACCUDROP:**

-   Remove rainbow beads from the sample port.

-   Set dial to RUN with no tube in place to rinse the up-take tubing.

-   Open the ACCUDROP experiment in the Browser window.

-   Load a tube of Accudrop beads.

-   RUN.

-   Turn off all external lights and get the room as dark as possible.

-   Run the beads at about 2-4000 events/sec.

-   Click Acquire on the software.

-   In the sort window, set the precision to Initial.

-   Put Accudrop filter in place by flipping the small silver bar on the
    side of the sort door.

-   Adjust the Drop Delay to optimize the stream on the left.

-   Set Sort Preciscion to Fine Tune

-   Adjust drop delay again, focusing on the center stream. Try to get
    the center (waste) stream as dim as possible.

**VANTAGE SHUT-DOWN:**

-   Remove sample tube.

-   Turn off the plate voltage.

-   Turn off the Drop Drive.

-   Install a tube of 10% bleach.

-   Turn Sample Differential all the way up.

-   Turn off the lasers at the back of the sorter.

-   Turn off the lasers at the Power Supply key.

-   Put the dial to STAND-BY and remove the bleach.

-   Turn the dial OFF.

-   Install a tube of dH2O.

-   Turn off the fluidics pressure switch.

-   Take the deflector plates off.

-   Put the pipette contraption (filled with dH2O) under the nozzle so
    that it is slightly submerged. Rest the bottom of the pipette on the
    waste trough. This is so that salt crystals do not dry on the
    nozzle.

-   Turn off the Drop Strobe and the Stream Lamps.

-   Turn off the house air.

-   Turn off the aerosol containment unit

-   Decontaminate all work surfaces with an appropriate disinfectant.

**PC:**

-   Quit the DIVA software.

-   Shut down the computer.

**5.2 Running a Sample**

**[VANTAGE SOP]{.underline}**

**[Running a Sample]{.underline}**

**[FACS Vantage SOP]{.underline}**

**[Running a Sample]{.underline}**

All samples to be run on the BD FACS Vantage must be in a single cell
suspension. Run cells through a 40um filter prior to arriving at the
sorter to achieve this if they are clumping together.

Only 5ml polystyrene round-bottom Falcon tubes can be inserted on the
up-take port. (Cat \# 352054).

-   Verify that the Whisper unit is turned on, that the filter life
    indicates 40% or more, and that the gauge is set to a level between
    the 2 black marks around the value 2. If any of these conditions
    cannot be achieved, stop and contact the facility manager. DO NOT
    CONTINUE!!!

-   Uncap sample tube and place cap on an alcohol pad.

-   Make sure the Vantage dial is set to [Stand-By]{.underline}.

-   Carefully slip the up-take tubing into the sample tube and lift the
    tube up towards the pressure seal.

-   Move the bottom arm beneath the tube. If necessary, adjust the
    height of the base by turning it.

```{=html}
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```
-   Set the dial to [Run]{.underline}.

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```
-   Press Boost to get the sample moving more quickly through the
    machine.

-   Once events are seen on the plots in the worksheet, you can make
    adjustments to voltages, compensation and threshold values.

-   When all settings are as you want them, click
    '[Record]{.underline}'.

-   Set the dial on the Vantage to '[Standby']{.underline} after the
    file has finished recording.

-   Gates can be set on the populations of interest in the software.

-   Choose '[Sort Layout]{.underline}' form the [Browser]{.underline}
    window, or open a previously used 'Sort Layout'.

-   Select the type of sort. (ie. 2-Tube/4-Tube/Standard slide etc).

-   Turn on the plate voltage by pressing the [Voltage]{.underline}
    button on the instrument control panel.

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```
-   Open the sort chamber door and place practice tubes or slide etc. in
    position.

-   Turn on '[Test Sort]{.underline}'.

-   In the software, click on the 'Streams' tab. Drag out the streams
    you wish to use by applying appropriate voltage to them.

> **[Note:]{.underline}** If only using 1 or 2 streams, use the 'left' &
> 'right' streams if using 3 or 4 streams, include the 'outer left' &
> 'outer right' streams. If using the plate-sorting option, use the
> 'outer left' stream only.

-   Apply the appropriate charge values to each stream in order to get
    the streams to aim for the center of their respective collection
    vessels. To see this, stand up and look down over the tops of the
    collection tubes to see the streams, (this will appear as a thin
    white line, like a strand of hair).

-   When the streams are correctly positioned, turn off the [Test
    Sort]{.underline}.

-   Remove the practice tubes or slide etc.

-   Replace with actual collection vessels, containing the appropriate
    collection media, if applicable.

```{=html}
<!-- -->
```
-   In the software, go to the Sort Layout window, for each stream,
    select a population to be sorted in that direction.

-   Turn the dial on the sorter to '[Run]{.underline}'.

-   Click 'Sort' on the sort layout window when you are ready to begin
    sorting.

-   If collection tubes need to be changed during the sort, turn the
    dial back to '[Stand-By']{.underline}. Open the chamber doors,
    replace tubes, close the chamber doors again. Then turn the dial
    back to '[Run]{.underline}' and resume sorting by clicking
    '[Sort]{.underline}' in the Sort Layout window.

-   At the end of the run, turn the dial to [Stand-By]{.underline}, cap
    & remove all collection tubes.

-   Proceed with the shut-down procedure.

**6.0 FACS Vantage Preventive Maintenance Procedures**

**[Precautions]{.underline}**

FACS Vantage Preventive Maintenance Procedure

Specific procedures for LBNL marked in RED.

Steps not applicable at LBNL have been crossed out and marked in RED.

# Objective

To provide a procedure and checklist for preventive maintenance (PM)
service call to be performed every six months. As part of the PM
procedures, a number of safety checks will be performed to ensure the
system meets the specifications for biological and laser safety as
required by LBNL.

# Purpose

The purpose of this program is to enhance the performance and improve
the reliability of the FACS Vantage instrument base by performing
routine checks, cleaning, and some parts replacements Estimate time to
complete: 4 hr's.

# Procedure

Prior to, and directly after, the PM, the Field Service Engineer (FSE)
is directed to perform resolution and sensitivity performance checks
with 2.49 m Nile Red Beads, sterile Chicken Red Blood Cells (cRBCs), and
CaliBRITE Beads, (use of Blue Beads and UV beads on an as required
basis). The intent of these checks is to document the instrument's
performance level prior to the PM, and any change to the performance
brought about by the PM.

# Pre PM

## Prior to the site visit, contact the customer and discuss the instrument's operation and performance. Determine if the instrument is in need of minor repair, if so, order the appropriate parts. If the instrument is in need of major repair (your judgment), advise the customer of the need for a repair visit prior to the PM. A PM will increase reliability and may enhance performance, it is not intended to be a substitute for a repair visit. Order the FACS Vantage PM kit (p/n 343640). {#prior-to-the-site-visit-contact-the-customer-and-discuss-the-instruments-operation-and-performance.-determine-if-the-instrument-is-in-need-of-minor-repair-if-so-order-the-appropriate-parts.-if-the-instrument-is-in-need-of-major-repair-your-judgment-advise-the-customer-of-the-need-for-a-repair-visit-prior-to-the-pm.-a-pm-will-increase-reliability-and-may-enhance-performance-it-is-not-intended-to-be-a-substitute-for-a-repair-visit.-order-the-facs-vantage-pm-kit-pn-343640. .unnumbered}

## Check the instrument's Field Change Notice (FCN) and Service Bulletin (SB) status. Order any parts required to bring the instrument up to current revision levels. {#check-the-instruments-field-change-notice-fcn-and-service-bulletin-sb-status.-order-any-parts-required-to-bring-the-instrument-up-to-current-revision-levels. .unnumbered}

## Upon arrival to site, align and operate the instrument and document (hardcopy) the performance level. {#upon-arrival-to-site-align-and-operate-the-instrument-and-document-hardcopy-the-performance-level. .unnumbered}

### Sensitivity Check: {#sensitivity-check .unnumbered}

#### Maximum separation of bimodal cRBC peak in the FSC detector. Dispose of remaining sample and any materials that have come in contact with sample into the LBNL-approved biohazard waste stream. {#maximum-separation-of-bimodal-crbc-peak-in-the-fsc-detector.-dispose-of-remaining-sample-and-any-materials-that-have-come-in-contact-with-sample-into-the-lbnl-approved-biohazard-waste-stream. .unnumbered}

### Resolution Check: {#resolution-check .unnumbered}

#### Minimum %CVs with 2.49 pm Nile Red beads in FSC, SSC, FLI, FL2, and FL3 detectors, Blue and UV beads in at least one detector if required equipment present. {#minimum-cvs-with-2.49-pm-nile-red-beads-in-fsc-ssc-fli-fl2-and-fl3-detectors-blue-and-uv-beads-in-at-least-one-detector-if-required-equipment-present. .unnumbered}

# Fluidic/Pneumatic System

## Reservoir Levels {#reservoir-levels .unnumbered}

### With the system pressurized, take note of the sheath and waste levels indicated on the video monitor. Depressurize the system. {#with-the-system-pressurized-take-note-of-the-sheath-and-waste-levels-indicated-on-the-video-monitor.-depressurize-the-system. .unnumbered}

### Check fluid levels in sheath and waste reservoirs, compare levels with the level indicated on the video screen. Calibrate if necessary. {#check-fluid-levels-in-sheath-and-waste-reservoirs-compare-levels-with-the-level-indicated-on-the-video-screen.-calibrate-if-necessary. .unnumbered}

## Remove sheath filter and prefilter, discard. {#remove-sheath-filter-and-prefilter-discard. .unnumbered}

### 343541 FILTER MILLIPORE (old Style) {#filter-millipore-old-style .unnumbered}

### 343542 SALINE FILTER CA 2503 (new style) {#saline-filter-ca-2503-new-style .unnumbered}

## Replace air filter {#replace-air-filter .unnumbered}

### 342832 FILTER ELEMENT-FOR REPLACEMENT ON 342831 342831 FILTER AIR 150PSI 1/8-NPT W/BOWL-GARD (listed for reference) {#filter-element-for-replacement-on-342831-342831-filter-air-150psi-18-npt-wbowl-gard-listed-for-reference .unnumbered}

## Monthly Cleaning {#monthly-cleaning .unnumbered}

Caution: Perform this procedure only upon customer approval as bleach
residues could cause customer applications problems.

### Perform 10% Household Bleach / 90% Distilled Water (DI) (freshly made) system flush. {#perform-10-household-bleach-90-distilled-water-di-freshly-made-system-flush. .unnumbered}

#### Empty waste reservoir and fill sheath reservoir approximately 1/4 full with DI water. Rinse the sheath reservoir thoroughly with DI water and remove all residual sheath solution. Empty fluid from sheath reservoir. {#empty-waste-reservoir-and-fill-sheath-reservoir-approximately-14-full-with-di-water.-rinse-the-sheath-reservoir-thoroughly-with-di-water-and-remove-all-residual-sheath-solution.-empty-fluid-from-sheath-reservoir. .unnumbered}

#### Fill the sheath reservoir approximately 1/4 full with bleach / DI solution, pressurize the system. {#fill-the-sheath-reservoir-approximately-14-full-with-bleach-di-solution-pressurize-the-system. .unnumbered}

#### Manipulate the fluidics control knob between FILL, OFF, and STANDBY until air is purged from the fluidic system. After the air is removed, set the control knob to each setting for sixty seconds. Repeat this cycle four times. Once cycled, set the fluidics control knob to the RUN position and allow the instrument to operate in this condition for 1/2 hour. This will allow the bleach to neutralize any bacteria present in the system. {#manipulate-the-fluidics-control-knob-between-fill-off-and-standby-until-air-is-purged-from-the-fluidic-system.-after-the-air-is-removed-set-the-control-knob-to-each-setting-for-sixty-seconds.-repeat-this-cycle-four-times.-once-cycled-set-the-fluidics-control-knob-to-the-run-position-and-allow-the-instrument-to-operate-in-this-condition-for-12-hour.-this-will-allow-the-bleach-to-neutralize-any-bacteria-present-in-the-system. .unnumbered}

#### Pour small quantity (.25 L) of bleach / DI solution into the sink assembly to clean the vacuum drain. {#pour-small-quantity-.25-l-of-bleach-di-solution-into-the-sink-assembly-to-clean-the-vacuum-drain. .unnumbered}

### Perform DI Water System Flush {#perform-di-water-system-flush .unnumbered}

#### Empty the sheath of bleach / DI solution. Fill the sheath reservoir 1/4 full with DI water. Rinse the inside of the sheath reservoir by shaking it thoroughly to remove all residual bleach / DI solution and empty. Fill the sheath reservoir approximately 1/4 full with DI water. {#empty-the-sheath-of-bleach-di-solution.-fill-the-sheath-reservoir-14-full-with-di-water.-rinse-the-inside-of-the-sheath-reservoir-by-shaking-it-thoroughly-to-remove-all-residual-bleach-di-solution-and-empty.-fill-the-sheath-reservoir-approximately-14-full-with-di-water. .unnumbered}

#### Manipulate the fluidic system control knob as in step (2.5.3) above, repeat this cycle twenty times to ensure all bleach / DI solution is purged from the fluidics system. Empty the sheath reservoir and fill it approximately 2/3 full with sheath fluid. Replace the sheath filter. {#manipulate-the-fluidic-system-control-knob-as-in-step-2.5.3-above-repeat-this-cycle-twenty-times-to-ensure-all-bleach-di-solution-is-purged-from-the-fluidics-system.-empty-the-sheath-reservoir-and-fill-it-approximately-23-full-with-sheath-fluid.-replace-the-sheath-filter. .unnumbered}

## Inspect all pneumatic lines feeding into the instrument for leaks. Secure with ty-wraps as needed. Replace / redress any discolored / kinked tubing. {#inspect-all-pneumatic-lines-feeding-into-the-instrument-for-leaks.-secure-with-ty-wraps-as-needed.-replace-redress-any-discolored-kinked-tubing. .unnumbered}

### 84-10003-00 MOUNT 2-WAY CABLE-TIE ADHESIVE BACKED {#mount-2-way-cable-tie-adhesive-backed .unnumbered}

### 84-10008-00 TIE WRAP 5.6\" long {#tie-wrap-5.6-long .unnumbered}

### 84-10019-00 TY WRAP 14 5/8\" long x 0.14\" width {#ty-wrap-14-58-long-x-0.14-width .unnumbered}

### 343690 TUBING SILASTIC-LAB.125\"ID X.25\" OD {#tubing-silastic-lab.125id-x.25-od .unnumbered}

## Open the Fluidics Control Panel and inspect all fluidic pneumatic lines for leaks, deformation, or discoloration. Pay particular attention to the valve switching area. Replace / redress as required. (FV/FSP only) {#open-the-fluidics-control-panel-and-inspect-all-fluidic-pneumatic-lines-for-leaks-deformation-or-discoloration.-pay-particular-attention-to-the-valve-switching-area.-replace-redress-as-required.-fvfsp-only .unnumbered}

### 343505 TUBING SILICONE 0.06 IN ID X 0.030 IN {#tubing-silicone-0.06-in-id-x-0.030-in .unnumbered}

### 343664 TUBING SILASTIC-LAB.062\"ID X.125\" OD {#tubing-silastic-lab.062id-x.125-od .unnumbered}

## Check sheath, sample, and bias pressure regulators for proper output. {#check-sheath-sample-and-bias-pressure-regulators-for-proper-output. .unnumbered}

### Sheath Regulator / +.15 to 30 psi\* (60 psi for FVSE) \*Limited by the amount of pressure from the source, usually 22 psi. (80 psi for FVSE) {#sheath-regulator-.15-to-30-psi-60-psi-for-fvse-limited-by-the-amount-of-pressure-from-the-source-usually-22-psi.-80-psi-for-fvse .unnumbered}

### Sample Regulator / -14 to +18 psi\*\* \*\* This regulator is capable of regulating vacuum, not applicable here. {#sample-regulator--14-to-18-psi-this-regulator-is-capable-of-regulating-vacuum-not-applicable-here. .unnumbered}

### Bias Regulator / 20 psi (Fixed) {#bias-regulator-20-psi-fixed .unnumbered}

## Check cam actuated leaf springs for proper pinch function. (FV/FSP only) {#check-cam-actuated-leaf-springs-for-proper-pinch-function.-fvfsp-only .unnumbered}

## Remove nozzle holder from the head drive, remove the nozzle tip and replace the nozzle holder o-ring. Reinstall nozzle tip onto nozzle holder and set aside. {#remove-nozzle-holder-from-the-head-drive-remove-the-nozzle-tip-and-replace-the-nozzle-holder-o-ring.-reinstall-nozzle-tip-onto-nozzle-holder-and-set-aside. .unnumbered}

### 343619 O-RING NOZZLE HOLDER {#o-ring-nozzle-holder .unnumbered}

## Remove and replace the three o-rings located between the sample tube (valve) clamp and the flow manifold. Apply a small amount of vacuum grease to each o-ring before replacement. {#remove-and-replace-the-three-o-rings-located-between-the-sample-tube-valve-clamp-and-the-flow-manifold.-apply-a-small-amount-of-vacuum-grease-to-each-o-ring-before-replacement. .unnumbered}

### 343625 O-RING SAMPLE AGITATOR (FV/FSP x2), (FVSE x3) {#o-ring-sample-agitator-fvfsp-x2-fvse-x3 .unnumbered}

### 343623 O-RING -- FACSTAR (FV/FSP only) {#o-ring-facstar-fvfsp-only .unnumbered}

### 347306 LUBRICANTO-RING SUPER-O-LUBE 1/2OZ TUBE {#lubricanto-ring-super-o-lube-12oz-tube .unnumbered}

## Replace the sample line and sample filter at this time, if required. Leave these assemblies behind if not used. Inspect the sample head area for air leaks using DI water placed on top of the knurled nut when running the instrument in run with a sample of DI water in a tube. Any air bubbles forming at the top of the nut should be addressed by tightening the nut slightly until the bubbles no longer form. {#replace-the-sample-line-and-sample-filter-at-this-time-if-required.-leave-these-assemblies-behind-if-not-used.-inspect-the-sample-head-area-for-air-leaks-using-di-water-placed-on-top-of-the-knurled-nut-when-running-the-instrument-in-run-with-a-sample-of-di-water-in-a-tube.-any-air-bubbles-forming-at-the-top-of-the-nut-should-be-addressed-by-tightening-the-nut-slightly-until-the-bubbles-no-longer-form. .unnumbered}

### 343501 LINE ASSEMBLY SAMPLE {#line-assembly-sample .unnumbered}

### 343647 FILTER SAMPLE SPARE {#filter-sample-spare .unnumbered}

## Replace the following spare parts as required {#replace-the-following-spare-parts-as-required .unnumbered}

### 343626 O-RING SAMPLE HEAD SM {#o-ring-sample-head-sm .unnumbered}

### 343618 O-RING FOR QUICK DISCONNECT {#o-ring-for-quick-disconnect .unnumbered}

### 343543 FILTER SINK FACS VANTAGE {#filter-sink-facs-vantage .unnumbered}

## Clean the outside of the sheath and waste tanks and inspect the inside of tanks for corrosion. Replace tank O-rings {#clean-the-outside-of-the-sheath-and-waste-tanks-and-inspect-the-inside-of-tanks-for-corrosion.-replace-tank-o-rings .unnumbered}

# Optical System

## Remove and clean all fluorescence optical elements, reinstall. {#remove-and-clean-all-fluorescence-optical-elements-reinstall. .unnumbered}

Note: Inspect band pass and dichroic filters for fingerprints and clean
if dirty using methanol and lens paper. These are coated lens so use as
little pressure as possible to avoid damaging the coating

### Collection Filters {#collection-filters .unnumbered}

### Beam Splitters {#beam-splitters .unnumbered}

### Field Lens {#field-lens .unnumbered}

## Inspect primary (1, 2), secondary (5, 6) and, if applicable, tertiary (7, 8) input prisms. Use dust chaser to remove any particulate. If prisms appear contaminated, clean with reagent grade methanol. {#inspect-primary-1-2-secondary-5-6-and-if-applicable-tertiary-7-8-input-prisms.-use-dust-chaser-to-remove-any-particulate.-if-prisms-appear-contaminated-clean-with-reagent-grade-methanol. .unnumbered}

## Inspect beam steering prisms (3, 4), use dust chaser to remove any particulate. If further cleaning is necessary, leave prisms mounted and utilize an orange stick with lens paper wrap to clean with reagent grade methanol. {#inspect-beam-steering-prisms-3-4-use-dust-chaser-to-remove-any-particulate.-if-further-cleaning-is-necessary-leave-prisms-mounted-and-utilize-an-orange-stick-with-lens-paper-wrap-to-clean-with-reagent-grade-methanol. .unnumbered}

## Inspect beam focusing lens. Use dust chaser to remove any particulate. Clean all elements if necessary. {#inspect-beam-focusing-lens.-use-dust-chaser-to-remove-any-particulate.-clean-all-elements-if-necessary. .unnumbered}

## Clean fluorescence objective face with cotton swab and DI water. {#clean-fluorescence-objective-face-with-cotton-swab-and-di-water. .unnumbered}

## Clean Stream Viewing Mirror. {#clean-stream-viewing-mirror. .unnumbered}

## Remove and clean the FSC collection lens. Replace the two sets screws securing the collection lens and front iris. {#remove-and-clean-the-fsc-collection-lens.-replace-the-two-sets-screws-securing-the-collection-lens-and-front-iris. .unnumbered}

### 74-30101-01 SCR 2-56X1/16 SOC ST CP C (x2) {#scr-2-56x116-soc-st-cp-c-x2 .unnumbered}

## Clean the two exposed optical surfaces mounted on the inside of the camera-viewing door (FV only) {#clean-the-two-exposed-optical-surfaces-mounted-on-the-inside-of-the-camera-viewing-door-fv-only .unnumbered}

## Clean the Accudrop camera optic if installed (FVSE only). {#clean-the-accudrop-camera-optic-if-installed-fvse-only. .unnumbered}

# Mechanical For the majority of mechanical checks listed below, the FSE is directed to attempt repair by making adjustments or mechanical repair. Assembly drawings of these mechanical subsystems are attached in order to assist in any on-site repair.

## Slides -- Rack and Pinion Type {#slides-rack-and-pinion-type .unnumbered}

### Check for smooth positive action, no rough spots, with minimum backlash. {#check-for-smooth-positive-action-no-rough-spots-with-minimum-backlash. .unnumbered}

#### Fluorescence objective focus slide {#fluorescence-objective-focus-slide .unnumbered}

#### Beam steering slides and rotational controls (qty 2) {#beam-steering-slides-and-rotational-controls-qty-2 .unnumbered}

#### Beam focus slide {#beam-focus-slide .unnumbered}

## Fluorescence Obscuration Holder Assembly {#fluorescence-obscuration-holder-assembly .unnumbered}

### Check fluorescence obscuration adjustments for smooth and full range movement of both vertical and rotation controls. Vacuum grease and included hardware should be used to repair any problems detected. {#check-fluorescence-obscuration-adjustments-for-smooth-and-full-range-movement-of-both-vertical-and-rotation-controls.-vacuum-grease-and-included-hardware-should-be-used-to-repair-any-problems-detected. .unnumbered}

## Forward Scatter Channel {#forward-scatter-channel .unnumbered}

### Check FSC obscuration adjustments for smooth and full range movement of both vertical and rotational controls. Use replacement hardware to repair any problems detected. {#check-fsc-obscuration-adjustments-for-smooth-and-full-range-movement-of-both-vertical-and-rotational-controls.-use-replacement-hardware-to-repair-any-problems-detected. .unnumbered}

### Check for proper retraction of FSC front collection barrel. {#check-for-proper-retraction-of-fsc-front-collection-barrel. .unnumbered}

### Check front and rear iris function. {#check-front-and-rear-iris-function. .unnumbered}

## Head (Stream) Adjust  {#head-stream-adjust .unnumbered}

### Check X, Y, and Z controls for smooth movement and adequate slide clearance. {#check-x-y-and-z-controls-for-smooth-movement-and-adequate-slide-clearance. .unnumbered}

### Check pitch controls for proper operation. (FV/FSP only) {#check-pitch-controls-for-proper-operation.-fvfsp-only .unnumbered}

## Wipe down the entire deflection illumination area, to include the deflection plates, with DI water. {#wipe-down-the-entire-deflection-illumination-area-to-include-the-deflection-plates-with-di-water. .unnumbered}

**Ventilation**

The stand-alone HEPA unit must be check to ensure it is operating
properly as specified by the manufacturer. Hoses will be checked for
leaks or damage and will be replaced as necessary. When necessary, the
HEPA filter will be replaced and disposed of as biohazardous waste. The
HEPA will then be leak tested and certified before the FACS unit is
placed back in service.

# Laser(s)

If open beam alignment is likely necessary (i.e. there are indications
that any of the lasers are significantly out of alignment, LBNL
personnel will alert the Lab's Laser Safety Officer (LSO) beforehand and
arrange for a Temporary Work Authorization (TWA) for the duration of the
open beam work. All controls and procedures in the TWA will be followed
without exception.

## Check and record output of all lasers at full power and primary operating wavelength. ~~Check UV output if the laser contains this option.~~ Check for proper mode. {#check-and-record-output-of-all-lasers-at-full-power-and-primary-operating-wavelength.-check-uv-output-if-the-laser-contains-this-option.-check-for-proper-mode. .unnumbered}

### Primary diode laser: 488nm, 200mW {#primary-diode-laser-488nm-200mw .unnumbered}

### ~~Dye head pumping laser: All lines~~ {#dye-head-pumping-laser-all-lines .unnumbered}

### ~~Krypton-ion laser: 407, 413, 567, 647nm~~ {#krypton-ion-laser-407-413-567-647nm .unnumbered}

### ~~Dye laser: 595nm~~ {#dye-laser-595nm .unnumbered}

### Red diode laser: 640nm, 60mW {#red-diode-laser-640nm-60mw .unnumbered}

### Violet diode laser: 405nm, 100mW {#violet-diode-laser-405nm-100mw .unnumbered}

### ~~Spectrum: 488, 567, 647 nm~~ {#spectrum-488-567-647-nm .unnumbered}

### ~~Enterprise 488nm & UV~~ {#enterprise-488nm-uv .unnumbered}

### Note: All Coherent output couplers are of a wedged shape design. This will cause the beam path to shift if the optic is rotated in relation to its holder. After the coupler has been removed from the laser, and previous to removing the optic from its holder, place a mark on the side wall of the optic and a corresponding mark on the orientation, minimizing alignment changes.

## Power down the laser(s). Clean all accessible optics to include Brewster windows. {#power-down-the-lasers.-clean-all-accessible-optics-to-include-brewster-windows. .unnumbered}

Note: Performed only for customers having a Laser Contract

## Check and record the tube voltage at maximum current of all water-cooled lasers. {#check-and-record-the-tube-voltage-at-maximum-current-of-all-water-cooled-lasers. .unnumbered}

## ~~Dye laser -- Clean any dye spills with paper towels and methanol.~~ {#dye-laser-clean-any-dye-spills-with-paper-towels-and-methanol. .unnumbered}

### ~~Dye pump~~ {#dye-pump .unnumbered}

### ~~Dye head~~ {#dye-head .unnumbered}

### ~~Optical bench~~ {#optical-bench .unnumbered}

## Realign and peak all lasers. Check and record output of all lasers at full power and primary operating wavelength. For lasers displaying improper mode, check again at this time for improvement. {#realign-and-peak-all-lasers.-check-and-record-output-of-all-lasers-at-full-power-and-primary-operating-wavelength.-for-lasers-displaying-improper-mode-check-again-at-this-time-for-improvement. .unnumbered}

### Primary diode laser: 488nm, 200mW {#primary-diode-laser-488nm-200mw-1 .unnumbered}

### ~~Dye head pumping laser: All lines~~ {#dye-head-pumping-laser-all-lines-1 .unnumbered}

### ~~Krypton-ion laser: 407, 413, 567, 647nm~~ {#krypton-ion-laser-407-413-567-647nm-1 .unnumbered}

### ~~Dye laser: 595nm~~ {#dye-laser-595nm-1 .unnumbered}

### Red diode laser: 640nm {#red-diode-laser-640nm .unnumbered}

### Violet diode laser: 405nm {#violet-diode-laser-405nm .unnumbered}

### ~~Spectrum: 488, 567, 647nm~~ {#spectrum-488-567-647nm .unnumbered}

### ~~Enterprise: 488nm & UV~~ {#enterprise-488nm-uv-1 .unnumbered}

## Check and record on the FSR the tube current and aperture setting at the standard operating wavelength and power. {#check-and-record-on-the-fsr-the-tube-current-and-aperture-setting-at-the-standard-operating-wavelength-and-power. .unnumbered}

# Electronics

## Measure and record on the FSR the line voltage, should be 120 VAC 10%. Check the Main AC Power Cord for excessive wear or fraying. Replace the power cord if necessary (#04-10547-00). {#measure-and-record-on-the-fsr-the-line-voltage-should-be-120-vac-10.-check-the-main-ac-power-cord-for-excessive-wear-or-fraying.-replace-the-power-cord-if-necessary-04-10547-00. .unnumbered}

### 04-10907-00 CABLE ASSY, POWER CORD (for 120 volts) (listed for reference) {#cable-assy-power-cord-for-120-volts-listed-for-reference .unnumbered}

### 04-10908-00 CABLE ASSY, POWER COR-INT (for 240 volts) (listed for reference) {#cable-assy-power-cor-int-for-240-volts-listed-for-reference .unnumbered}

## Check +5V Digital, +15V and --15 Analog low voltage power supplies on the Log Amp PCB by mounting the board on an extender board and probing the edge connectors. Check the +15V Digital on the Deflection Illumination Board. {#check-5v-digital-15v-and-15-analog-low-voltage-power-supplies-on-the-log-amp-pcb-by-mounting-the-board-on-an-extender-board-and-probing-the-edge-connectors.-check-the-15v-digital-on-the-deflection-illumination-board. .unnumbered}

### Log Amp PCB: {#log-amp-pcb .unnumbered}

#### Analog Ground: Pin 42 {#analog-ground-pin-42 .unnumbered}

#### Digital Ground: Pin 1 {#digital-ground-pin-1 .unnumbered}

#### +5V Digital: Pin 93 (Acceptable +5.0 to 5.10) {#v-digital-pin-93-acceptable-5.0-to-5.10 .unnumbered}

#### +15V Analog: Pin 39 (Acceptable +15.0 to 15.3) {#v-analog-pin-39-acceptable-15.0-to-15.3 .unnumbered}

#### -15V Analog: Pin 37 (Acceptable -15.0 to -15.3) {#v-analog-pin-37-acceptable--15.0-to--15.3 .unnumbered}

### Deflection Illumination PCB: {#deflection-illumination-pcb .unnumbered}

#### +5V Digital: U4, Pin 16 (Acceptable +5.0 to 5.10) {#v-digital-u4-pin-16-acceptable-5.0-to-5.10 .unnumbered}

## Card Cage Check for operation and function of user adjustments. {#card-cage-check-for-operation-and-function-of-user-adjustments. .unnumbered}

### P1 -- P8 Log Offset -- will shift a population approximately one log decade. (Card 14, 15) {#p1-p8-log-offset-will-shift-a-population-approximately-one-log-decade.-card-14-15 .unnumbered}

### Dead Time Adjust -- lengthens intensified region on pulse display, normally CCW, minimum dead time length, (use CellQuest on the FVSE). {#dead-time-adjust-lengthens-intensified-region-on-pulse-display-normally-ccw-minimum-dead-time-length-use-cellquest-on-the-fvse. .unnumbered}

### Dual Laser Delay Adjust -- controls the time delay of second laser signals relative to the first, , (use CellQuest on the FVSE). {#dual-laser-delay-adjust-controls-the-time-delay-of-second-laser-signals-relative-to-the-first-use-cellquest-on-the-fvse. .unnumbered}

### Test Pulse Rate -- controls the frequency at which test signals are synchronously generated in the eight detector channels. (Card 8) {#test-pulse-rate-controls-the-frequency-at-which-test-signals-are-synchronously-generated-in-the-eight-detector-channels.-card-8 .unnumbered}

### Test Pulse Amplitude (six Controls) -- controls the amplitude of the eight detector test pulse generators. {#test-pulse-amplitude-six-controls-controls-the-amplitude-of-the-eight-detector-test-pulse-generators. .unnumbered}

### Test Signal Noise -- controls the level of noise superimposed on top of the two test populations. (Card 8). {#test-signal-noise-controls-the-level-of-noise-superimposed-on-top-of-the-two-test-populations.-card-8. .unnumbered}

### Drop Sync Switch -- synchronizes test signal generation with the drop dire generator. (Card 2 FVSE; Card 6 FV/FSP) {#drop-sync-switch-synchronizes-test-signal-generation-with-the-drop-dire-generator.-card-2-fvse-card-6-fvfsp .unnumbered}

## Main Control Panel Check for proper operation of the following controls (For DiVa option use appropriate controls): {#main-control-panel-check-for-proper-operation-of-the-following-controls-for-diva-option-use-appropriate-controls .unnumbered}

### Left, right, and center stream controls {#left-right-and-center-stream-controls .unnumbered}

### Phase control {#phase-control .unnumbered}

### Amplitude control {#amplitude-control .unnumbered}

### Deflection power {#deflection-power .unnumbered}

### Droplet strobe switch {#droplet-strobe-switch .unnumbered}

### Drop drive frequency control {#drop-drive-frequency-control .unnumbered}

### Stream illumination switch {#stream-illumination-switch .unnumbered}

## Log Amp Check {#log-amp-check .unnumbered}

### Perform for each fluorescence parameter. Position a test signal at channel 80 in linear gain. Set gain to log and record channel number (x). Reset to a linear gain and position the test signal at channel 800. Set gain to log and record channel number (y). For field purpose of this procedure is to identify log amplifiers that have been tampered with and are therefore grossly out of calibration. This test is not intended to check calibration with the accuracy of that accomplished in manufacturing with test fixtures. {#perform-for-each-fluorescence-parameter.-position-a-test-signal-at-channel-80-in-linear-gain.-set-gain-to-log-and-record-channel-number-x.-reset-to-a-linear-gain-and-position-the-test-signal-at-channel-800.-set-gain-to-log-and-record-channel-number-y.-for-field-purpose-of-this-procedure-is-to-identify-log-amplifiers-that-have-been-tampered-with-and-are-therefore-grossly-out-of-calibration.-this-test-is-not-intended-to-check-calibration-with-the-accuracy-of-that-accomplished-in-manufacturing-with-test-fixtures. .unnumbered}

# Computer

## Clean CRT screen(s) {#clean-crt-screens .unnumbered}

## Clean printer(s) and verify proper operation utilizing self test diagnostics. {#clean-printers-and-verify-proper-operation-utilizing-self-test-diagnostics. .unnumbered}

## Verify read/write operation of hard disk drive(s). {#verify-readwrite-operation-of-hard-disk-drives. .unnumbered}

## Check for proper keyboard function. {#check-for-proper-keyboard-function. .unnumbered}

## Clean the ball on mechanical mice. {#clean-the-ball-on-mechanical-mice. .unnumbered}

# Overall Cleaning and check of biosafety provisions.

## With a DI soaked towel or commercial cleaner, wipe down all exterior surfaces. {#with-a-di-soaked-towel-or-commercial-cleaner-wipe-down-all-exterior-surfaces. .unnumbered}

## Verify that the Aerosol Management Option (AMO) is operating within specifications using the AMO Field Service Kit. {#verify-that-the-aerosol-management-option-amo-is-operating-within-specifications-using-the-amo-field-service-kit. .unnumbered}

## Check the filter integrity gage located on the Whisper vacuum pump. If integrity reads 40% or below, direct LBNL personnel to order a replacement filter kit, part no. 333030. {#check-the-filter-integrity-gage-located-on-the-whisper-vacuum-pump.-if-integrity-reads-40-or-below-direct-lbnl-personnel-to-order-a-replacement-filter-kit-part-no.-333030. .unnumbered}

### If a new filter is needed, LBNL personnel will order a new filter kit. {#if-a-new-filter-is-needed-lbnl-personnel-will-order-a-new-filter-kit. .unnumbered}

### LBNL personnel will schedule the Lab's contractor (currently TSS) to verify the proper functioning of the filter kit. {#lbnl-personnel-will-schedule-the-labs-contractor-currently-tss-to-verify-the-proper-functioning-of-the-filter-kit. .unnumbered}

### LBNL personnel will install the new filter kit and dispose of the used kit in the biohazard waste. {#lbnl-personnel-will-install-the-new-filter-kit-and-dispose-of-the-used-kit-in-the-biohazard-waste. .unnumbered}

# Post PM

## Power up the instrument, realign optical system. {#power-up-the-instrument-realign-optical-system. .unnumbered}

## Run and document performance checks as in Pre PM. {#run-and-document-performance-checks-as-in-pre-pm. .unnumbered}

During sort check, run sort profile, determine drop delay offset, if any
adjust offset on Sort Control PCB if needed, follow the general
guidelines below.

### Run 2 um Nile red beads and acquire histograms for primary laser. Record the results on the FSR {#run-2-um-nile-red-beads-and-acquire-histograms-for-primary-laser.-record-the-results-on-the-fsr .unnumbered}

### If present, run UV alignment beads for secondary or tertiary laser alignment of UV laser. Record the results on the FSR {#if-present-run-uv-alignment-beads-for-secondary-or-tertiary-laser-alignment-of-uv-laser.-record-the-results-on-the-fsr .unnumbered}

### If present, run Blue beads for secondary or tertiary laser alignment of red laser. Record the results on the FSR {#if-present-run-blue-beads-for-secondary-or-tertiary-laser-alignment-of-red-laser.-record-the-results-on-the-fsr .unnumbered}

### Run CRBCs and acquire a histogram for FSC. Record the ratio of the two peaks on the FSR. {#run-crbcs-and-acquire-a-histogram-for-fsc.-record-the-ratio-of-the-two-peaks-on-the-fsr. .unnumbered}

### Using Calibrite beads if available or another bead mixture perform a sort at the pressure most often used by the customer. Attach a print out of the results with the FSR and record the purity on the FSR. {#using-calibrite-beads-if-available-or-another-bead-mixture-perform-a-sort-at-the-pressure-most-often-used-by-the-customer.-attach-a-print-out-of-the-results-with-the-fsr-and-record-the-purity-on-the-fsr. .unnumbered}

# End Of Procedure 

# Appendix A: LBNL -- Life Science Divison Flow Facility

# FACS User Log

<table>
<colgroup>
<col style="width: 6%" />
<col style="width: 6%" />
<col style="width: 12%" />
<col style="width: 13%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 10%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 6%" />
<col style="width: 8%" />
</colgroup>
<tbody>
<tr class="odd">
<td><h1 id="date">Date</h1></td>
<td><h1 id="time">Time</h1></td>
<td><h1 id="users-name">Users Name</h1></td>
<td><h1 id="users-signature">Users Signature</h1></td>
<td><h1 id="pi">PI</h1></td>
<td><h1 id="e-mail">e-mail</h1></td>
<td><h1 id="sample-description">Sample Description</h1></td>
<td><h1 id="potential-hazards">Potential Hazards</h1></td>
<td><h1 id="ehs-training-com-pleted">EH&amp;S Training
Com-pleted</h1></td>
<td><h1 id="bua-com-pleted">BUA<br />
Com-pleted</h1></td>
<td><h1 id="facs-training-level">FACS Training Level</h1></td>
</tr>
<tr class="even">
<td><h1 id="section-1"></h1></td>
<td><h1 id="section-2"></h1></td>
<td><h1 id="section-3"></h1></td>
<td><h1 id="section-4"></h1></td>
<td><h1 id="section-5"></h1></td>
<td><h1 id="section-6"></h1></td>
<td><h1 id="section-7"></h1></td>
<td><h1 id="section-8"></h1></td>
<td><h1 id="section-9"></h1></td>
<td><h1 id="section-10"></h1></td>
<td><h1 id="section-11"></h1></td>
</tr>
<tr class="odd">
<td><h1 id="section-12"></h1></td>
<td><h1 id="section-13"></h1></td>
<td><h1 id="section-14"></h1></td>
<td><h1 id="section-15"></h1></td>
<td><h1 id="section-16"></h1></td>
<td><h1 id="section-17"></h1></td>
<td><h1 id="section-18"></h1></td>
<td><h1 id="section-19"></h1></td>
<td><h1 id="section-20"></h1></td>
<td><h1 id="section-21"></h1></td>
<td><h1 id="section-22"></h1></td>
</tr>
<tr class="even">
<td><h1 id="section-23"></h1></td>
<td><h1 id="section-24"></h1></td>
<td><h1 id="section-25"></h1></td>
<td><h1 id="section-26"></h1></td>
<td><h1 id="section-27"></h1></td>
<td><h1 id="section-28"></h1></td>
<td><h1 id="section-29"></h1></td>
<td><h1 id="section-30"></h1></td>
<td><h1 id="section-31"></h1></td>
<td><h1 id="section-32"></h1></td>
<td><h1 id="section-33"></h1></td>
</tr>
<tr class="odd">
<td><h1 id="section-34"></h1></td>
<td><h1 id="section-35"></h1></td>
<td><h1 id="section-36"></h1></td>
<td><h1 id="section-37"></h1></td>
<td><h1 id="section-38"></h1></td>
<td><h1 id="section-39"></h1></td>
<td><h1 id="section-40"></h1></td>
<td><h1 id="section-41"></h1></td>
<td><h1 id="section-42"></h1></td>
<td><h1 id="section-43"></h1></td>
<td><h1 id="section-44"></h1></td>
</tr>
<tr class="even">
<td><h1 id="section-45"></h1></td>
<td><h1 id="section-46"></h1></td>
<td><h1 id="section-47"></h1></td>
<td><h1 id="section-48"></h1></td>
<td><h1 id="section-49"></h1></td>
<td><h1 id="section-50"></h1></td>
<td><h1 id="section-51"></h1></td>
<td><h1 id="section-52"></h1></td>
<td><h1 id="section-53"></h1></td>
<td><h1 id="section-54"></h1></td>
<td><h1 id="section-55"></h1></td>
</tr>
<tr class="odd">
<td><h1 id="section-56"></h1></td>
<td><h1 id="section-57"></h1></td>
<td><h1 id="section-58"></h1></td>
<td><h1 id="section-59"></h1></td>
<td><h1 id="section-60"></h1></td>
<td><h1 id="section-61"></h1></td>
<td><h1 id="section-62"></h1></td>
<td><h1 id="section-63"></h1></td>
<td><h1 id="section-64"></h1></td>
<td><h1 id="section-65"></h1></td>
<td><h1 id="section-66"></h1></td>
</tr>
<tr class="even">
<td><h1 id="section-67"></h1></td>
<td><h1 id="section-68"></h1></td>
<td><h1 id="section-69"></h1></td>
<td><h1 id="section-70"></h1></td>
<td><h1 id="section-71"></h1></td>
<td><h1 id="section-72"></h1></td>
<td><h1 id="section-73"></h1></td>
<td><h1 id="section-74"></h1></td>
<td><h1 id="section-75"></h1></td>
<td><h1 id="section-76"></h1></td>
<td><h1 id="section-77"></h1></td>
</tr>
<tr class="odd">
<td><h1 id="section-78"></h1></td>
<td><h1 id="section-79"></h1></td>
<td><h1 id="section-80"></h1></td>
<td><h1 id="section-81"></h1></td>
<td><h1 id="section-82"></h1></td>
<td><h1 id="section-83"></h1></td>
<td><h1 id="section-84"></h1></td>
<td><h1 id="section-85"></h1></td>
<td><h1 id="section-86"></h1></td>
<td><h1 id="section-87"></h1></td>
<td><h1 id="section-88"></h1></td>
</tr>
<tr class="even">
<td><h1 id="section-89"></h1></td>
<td><h1 id="section-90"></h1></td>
<td><h1 id="section-91"></h1></td>
<td><h1 id="section-92"></h1></td>
<td><h1 id="section-93"></h1></td>
<td><h1 id="section-94"></h1></td>
<td><h1 id="section-95"></h1></td>
<td><h1 id="section-96"></h1></td>
<td><h1 id="section-97"></h1></td>
<td><h1 id="section-98"></h1></td>
<td><h1 id="section-99"></h1></td>
</tr>
<tr class="odd">
<td><h1 id="section-100"></h1></td>
<td><h1 id="section-101"></h1></td>
<td><h1 id="section-102"></h1></td>
<td><h1 id="section-103"></h1></td>
<td><h1 id="section-104"></h1></td>
<td><h1 id="section-105"></h1></td>
<td><h1 id="section-106"></h1></td>
<td><h1 id="section-107"></h1></td>
<td><h1 id="section-108"></h1></td>
<td><h1 id="section-109"></h1></td>
<td><h1 id="section-110"></h1></td>
</tr>
<tr class="even">
<td><h1 id="section-111"></h1></td>
<td><h1 id="section-112"></h1></td>
<td><h1 id="section-113"></h1></td>
<td><h1 id="section-114"></h1></td>
<td><h1 id="section-115"></h1></td>
<td><h1 id="section-116"></h1></td>
<td><h1 id="section-117"></h1></td>
<td><h1 id="section-118"></h1></td>
<td><h1 id="section-119"></h1></td>
<td><h1 id="section-120"></h1></td>
<td><h1 id="section-121"></h1></td>
</tr>
<tr class="odd">
<td><h1 id="section-122"></h1></td>
<td><h1 id="section-123"></h1></td>
<td><h1 id="section-124"></h1></td>
<td><h1 id="section-125"></h1></td>
<td><h1 id="section-126"></h1></td>
<td><h1 id="section-127"></h1></td>
<td><h1 id="section-128"></h1></td>
<td><h1 id="section-129"></h1></td>
<td><h1 id="section-130"></h1></td>
<td><h1 id="section-131"></h1></td>
<td><h1 id="section-132"></h1></td>
</tr>
<tr class="even">
<td><h1 id="section-133"></h1></td>
<td><h1 id="section-134"></h1></td>
<td><h1 id="section-135"></h1></td>
<td><h1 id="section-136"></h1></td>
<td><h1 id="section-137"></h1></td>
<td><h1 id="section-138"></h1></td>
<td><h1 id="section-139"></h1></td>
<td><h1 id="section-140"></h1></td>
<td><h1 id="section-141"></h1></td>
<td><h1 id="section-142"></h1></td>
<td><h1 id="section-143"></h1></td>
</tr>
<tr class="odd">
<td><h1 id="section-144"></h1></td>
<td><h1 id="section-145"></h1></td>
<td><h1 id="section-146"></h1></td>
<td><h1 id="section-147"></h1></td>
<td><h1 id="section-148"></h1></td>
<td><h1 id="section-149"></h1></td>
<td><h1 id="section-150"></h1></td>
<td><h1 id="section-151"></h1></td>
<td><h1 id="section-152"></h1></td>
<td><h1 id="section-153"></h1></td>
<td><h1 id="section-154"></h1></td>
</tr>
<tr class="even">
<td><h1 id="section-155"></h1></td>
<td><h1 id="section-156"></h1></td>
<td><h1 id="section-157"></h1></td>
<td><h1 id="section-158"></h1></td>
<td><h1 id="section-159"></h1></td>
<td><h1 id="section-160"></h1></td>
<td><h1 id="section-161"></h1></td>
<td><h1 id="section-162"></h1></td>
<td><h1 id="section-163"></h1></td>
<td><h1 id="section-164"></h1></td>
<td><h1 id="section-165"></h1></td>
</tr>
<tr class="odd">
<td><h1 id="section-166"></h1></td>
<td><h1 id="section-167"></h1></td>
<td><h1 id="section-168"></h1></td>
<td><h1 id="section-169"></h1></td>
<td><h1 id="section-170"></h1></td>
<td><h1 id="section-171"></h1></td>
<td><h1 id="section-172"></h1></td>
<td><h1 id="section-173"></h1></td>
<td><h1 id="section-174"></h1></td>
<td><h1 id="section-175"></h1></td>
<td><h1 id="section-176"></h1></td>
</tr>
<tr class="even">
<td><h1 id="section-177"></h1></td>
<td><h1 id="section-178"></h1></td>
<td><h1 id="section-179"></h1></td>
<td><h1 id="section-180"></h1></td>
<td><h1 id="section-181"></h1></td>
<td><h1 id="section-182"></h1></td>
<td><h1 id="section-183"></h1></td>
<td><h1 id="section-184"></h1></td>
<td><h1 id="section-185"></h1></td>
<td><h1 id="section-186"></h1></td>
<td><h1 id="section-187"></h1></td>
</tr>
</tbody>
</table>

#   {#section-188}

# Appendix B: FSE Tools/Supplies Required

1)  342868 Chicken Red Blood Cells (cRBCs)

2)  347240 2.49 m Nile Red Beads

3)  347241 6.0 m Blue Beads

4)  344669 2.5 m UV Beads

5)  340486 Three Color CaliBRITE Beads

6)  340487 APC CaliBRITE Beads

7)  349523 DNA QC Kit

8)  343675 Falcon 12 X 75 Tubes

9)  Extender Board

10) Laser Power Meter

11) Air Pressure Gauge

12) Digital Volt Meter

# Customer Supplies Required

1)  DI Water (Double Glass Distilled)

2)  Bleach (Clorox)

3)  Vortex or Sonication Bath

4)  Reagent Grade Methanol

5)  Paper Towls

6)  Rubber Gloves

7)  Safety Glasses

8)  Lab Coat

9)  Floppy (and Optional Removable Media) Drive Head Cleaning Kit

10) CRT Cleaner

11) Commercial Cleaner

# PM Kit FS/FSP & FV/FVSE (p/n 343640) PartsList

\|\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--\|

\|Material 343640 D 0003 Alt. Usage 1 \|

\| PM KIT FS/FSP & FV/FVSE Valid 12/21/1999\|

\|Reqd qty 1.000 Base quant 1.000 EA \|

\|\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--\|

\|Lv Item Component no. Quant Un Ict Asm\|

\| Description Ex. \|

\|\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--\|

\| 1 0010 343647 C 1.000 EA L \|

\| FILTER SAMPLE SPARE \|

\| 1 0020 343501 A 1.000 EA L \|

\| LINE ASSEMBLY SAMPLE \|

\| 1 0030 343541 A 10.000 EA L \|

\| FILTER MILLIPORE \|

\| 1 0040 59-10027-00 B 2.000 EA L \|

\| FITTING, \"T\" 1/16 ID \|

\| 1 0050 59-10056-00 A 4.000 EA L \|

\| FITTING ELBOW (L10-1) \|

\| 1 0060 74-30101-01 2.000 EA L \|

\| SCR 2-56X1/16 SOC ST CP C \|

\| 1 0070 343664 B 50.000 IN L \|

\| TUBING SILASTIC-LAB .062\"ID X .125\" OD \|

\| 1 0080 343690 B 50.000 IN L \|

\| TUBING SILASTIC-LAB .125\"ID X .25\" OD \|

\| 1 0090 343505 A 50.000 IN L \|

\| TUBING SILICONE 0.06 IN ID X 0.030 IN \|

\| 1 0100 343619 A 1.000 EA L \|

\| O-RING NOZZLE HOLDER \|

\| 1 0110 343710 A 2.000 EA L \|

\| CONN,FEM LUER TO 10-32 \|

\| 1 0120 343568 A 2.000 EA L \|

\| FIT, 1/8 MALE \|

\| 1 0130 343623 A 3.000 EA L \|

\| O-RING - FACSTAR \|

\| 1 0140 343625 A 2.000 EA L \|

\| O-RING SAMPLE AGITATOR \|

\| 1 0150 88-20138-00 A 3.000 EA L \|

\| WASHER, RUBBER SEAL W/FILTER \|

\| 1 0160 343626 A 1.000 EA L \|

\| O-RING SAMPLE HEAD SM \|

\| 1 0170 343618 A 5.000 EA L \|

\| O-RING FOR QUICK DISCONNECT \|

\| 1 0180 343680 A 1.000 EA L \|

\| STOPCOCK 3 WAY \|

\| 1 0190 343542 A 1.000 EA L \|

\| SALINE FILTER CA 2503 \|

\| 1 0200 343538 A 2.000 EA L \|

\| FLTR AIR HYDROPHOBIC WHITE 1-1/4 IN DIA \|

\| 1 0210 343543 A 1.000 EA L \|

\| FILTER SINK FACS VANTAGE \|

\| 1 0220 343546 A 1.000 EA L \|

\| FILTER VAC SHIELD 3INDIA/REPL 2-1/4INDIA \|

\|\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--\|

\|\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--\|

\|Material 343640 D 0003 Alt. Usage 1 \|

\| PM KIT FS/FSP & FV/FVSE Valid 12/21/1999\|

\|Reqd qty 1.000 Base quant 1.000 EA \|

\|\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--\|

\|Lv Item Component no. Quant Un Ict Asm\|

\| Description Ex. \|

\|\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--\|

\| 1 0230 343531 A 1.000 EA L \|

\| COUPLING INS 1/4 IN NPT \|

\| 1 0240 347306 A 1.000 EA L \|

\| LUBRICANTO-RING SUPER-O-LUBE 1/2OZ TUBE \|

\| 1 0250 347308 A 1.000 EA L \|

\| TISSUE LENS CLEANER 4\" X6\"50-SHT BOOKLET \|

\| 1 0260 99-30122-00 B 1.000 EA L \|

\| APPLICATOR, COTTON TIPPED \|

\| 1 0270 59-10084-00 A 1.000 EA L \|

\| ELBOW BARB FITTING 10-32 \|

\| 1 0280 84-10008-00 A 10.000 EA L \|

\| TIE WRAP \|

\| 1 0290 84-10019-00 A 5.000 EA L \|

\| TY WRAP \|

\| 1 0300 88-20149-00 A 2.000 EA L \|

\| O-RING 3.475 X .275 \|

\| 1 0310 84-10003-00 B 3.000 EA L \|

\| MOUNT 2-WAY CABLE-TIE ADHESIVE BACKED \|

\| 1 0320 59-10057-00 01 4.000 EA L \|

\| PORT TUBE (NYLON) - FACSTAR \|

\| 1 0330 59-10200-00 A 1.000 EA L \|

\| 1/4 FLOW INSERT 1/4\" HOSE \|

\| 1 0340 59-10198-00 A 1.000 EA L \|

\| 1/4 COUPLING 1/4 HOSE & PANEL \|

\| 1 0350 59-10194-00 A 1.000 EA L \|

\| 1/4\"COUPLING 1/4\" HOSE \|

\| 1 0360 59-10199-00 A 1.000 EA L \|

\| 1/4 INSERT 1/4 HOSE PANEL \|

\| 1 0370 342832 A 2.000 EA L \|

\| FILTER ELEMENT-FOR REPLACEMENT ON 342831 \|

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

# FACS Vantage

# Preventive Maintenance

**Checklist**

FLUIDIC/OPTICAL ( ) Clean Stream View Mirror ELECTRONIC/COMPUTER

( ) Clean FSC Collec. Lens

( ) Check Sheath/Waste Lvl ( ) Clean Camera Optics ( ) Check Line
Voltage

Detectos / Calibrate ( ) Measure DC Power Supplies

( ) Replace Air Filter MECHANICAL/LASERS ( ) Check PCB Card Controls

( ) DI/Bleach Flush ( ) Check PMT Power Supplies

( ) DI Flush/Chg Sheath Filter ( ) Check Fluor. Focus Slide ( ) Check
Main Control Panel

( ) Check Tubing/Fittings ( ) Check Beam Steer. Slides ( ) Log Amp Check

( ) Check Regulators ( ) Check Beam Steer. Rotate ( ) Clean CRT & Floppy
Drive

( ) Check Leaf Springs ( ) Check Beam Focus Slide ( ) Clean/Check
Printer

( ) Replace Nozzle O-ring ( ) Check Fluor. Obscuration ( ) Check
Read/Write Floppy Drive

( ) Replace 3 Sample Manifold ( ) Check FSC Obscuration ( ) Check
Read/Write Hard Drive

O-rings ( ) Check FSC Iris (qty 2) ( ) Check Keyboard

( ) Replace Sample Line/Filter ( ) Check Stream Adjust Assy.

( ) Clean Fluor. Optics ( ) Clean Defl. & Illum. Area PERFORMANCE CHECK

( ) Inspect/Clean Input Prisms ( ) Check Laser(s) Power

( ) Inspect/Clean Steer. Prisms ( ) Check Laser Optics ( )
Sensitivity/Resolution

( ) Inspect Beam Focus Lens ( ) Check Tube(s) Voltage ( ) Sorting

( ) Clean Fluor. Objective ( ) Check Laser(s) Power ( ) Hard Copy
Attached

**History of Changes**

Changes from original version to documented version p/n 33xxxx Rev. A

1.  Provide sequential numbering to document.

2.  Procedure: Remove 2.0μm beads, replace with 2.49μm Nile Red Beads.

3.  Procedure: Added, "use Blue Beads and UV Beads as required".

4.  Pre PM: Changed PM Kit part number from p/n 12-00307-00 to p/n
    343640.

5.  Pre PM Resolution Check was:\
    Minimum %CV with 2 micron green beads in FSC and FL2 detectors.
    Changed to:\
    Minimum %CV with 2.49μm Nile Red beads in FSC, SSC, FL1, FL2 and FL3
    detectors, Blue Beads and UV in at least one detector if required
    equipment present.

6.  Fluidic/Pneumatic System, step 3: Add part number for new sheath
    filter (p/n 343542) and old sheath filter element (p/n 343541).

7.  Fluidic/Pneumatic System, step 4: Remove obsolete air filter (p/n
    41-10001-00) replace with Filter Element (p/n 342832) for use on the
    FVSE.

8.  Perform DI water system flush B; was: as in step 6C above, should
    be: as in step 5C.

9.  Fluidic/Pneumatic System, step 7: Ty Wrap 8 1/8" long x 0.14" width
    (p/n 84-10001-00) was removed from the PM kit, use Ty Wrap 5.6" long
    (p/n 84-10008-00) or Ty Wrap 14 5/8" long x 0.14" width (p/n
    84-10019-00).

10. Fluidic/Pneumatic System, step 7: Tubing Silicone .125IDX.250 OD
    (p/n 80-10011-00) is obsolete, replaced with Tubing Silastic-Lab
    .125\"ID X .25\" OD (p/n 343690).

11. Fluidic/Pneumatic System, step 8: Tubing 1/16X1/32 (p/n 80-30005-00)
    is obsolete, replaced with Tubing Silicone 0.06 IN ID X 0.030 IN
    (p/n 343505).

12. Fluidic/Pneumatic System, step 8: Tubing Silicone.062IDX.125 OD (p/n
    80-10005-00) was replaced in the kit by Tubing Silastic-Lab .062\"ID
    X .125\" OD (p/n 343664).

13. Fluidic/Pneumatic System, step 9: Updated to reflect FVSE pressure
    requirements.

14. Fluidic/Pneumatic System, step 11: O-Ring .146IDx.032W 70BN is
    obsolete, replaced with O-Ring Nozzle Holder (p/n 343619).

15. Fluidic/Pneumatic System, step 12: O-Ring Buna-N Parker 2-006 --FS
    (p/n 88-20058-00) is obsolete replaced with O-Ring Sample Agitator
    (p/n 343625).

16. Fluidic/Pneumatic System, step 12: O-Ring - FACStar (p/n
    88-20056-00) changed part number, replaced with O-Ring - FACStar
    (p/n 343623).

17. Fluidic/Pneumatic System, step 13: Sample Line Assy, .010 (p/n
    02-61358-00) changed part number, replaced with Line Assembly Sample
    (p/n 343501).

18. Fluidic/Pneumatic System, step 13: Assy, Sample Filter S/D/L ANL
    SENS (p/n 02-30726-00) changed part number, replaced with Filter
    Sample Spare (p/n 343647).

19. Mechanical, step 2, Fluorescence Obscuration Holder Assembly:
    Compression Spring (S.STL)-FS (p/n 78-10040-00) and Spring,
    Extension (p/n 78-10055-00) were removed from the kit.

20. Mechanical, step 3, Forward Scatter Channel: Spring Extension 3/4 IN
    (p/n 78-10006-00) and Spring (p/n 78-10008-00) were removed from the
    kit.

21. Electronics, step 1: Cable Assy, Power Cord (p/n 04-10547-00) is
    obsolete, replaced with Cable Assy, Power Cord (p/n 04-10907-00) for
    120 volts. (p/n 04-10908-00) for 240 volts.

22. Electronics, step 3, Card Cage, B and c, Added "use CellQuest on the
    FVSE"

23. Electronics, step 5, updated for DiVa option.

24. Updated Tools/Supplies Required.

25. Remove (1.3.1.1 Maximum separation of unlabeled CaliBRITE Beads and
    cRBCs in all fluorescence detectors. PMTs set on unlabeled beads,
    compensation at %0.00.) as FSEs do not carry CaliBRITE Beads.

26. Monthly cleaning performed only upon customer approval.

27. Added (2.11. Inspect the sample head area for air leaks using DI
    water placed on top of the knurled nut when running the instrument
    in run with a sample of DI water in a tube. Any air bubbles forming
    at the top of the nut should be addressed by tightening the nut
    slightly until the bubbles no longer form.)

28. Added (2.13 Clean the outside of the sheath and waste tanks and
    inspect the inside of tanks for corrosion. Replace tank O-rings).

29. Note on 3.1

30. Added (3.9 Clean the Accudrop camera optic if installed (FVSE
    only).)

31. Added Note on 5.2.

32. Removed: (6.4 PMT Power supplies)

33. Removed: activities with floppy drive.

34. Added 9.2.1 to 9.2.5

35. Added LBNL-specific safety steps