Single-Cell Analysis

Celselect Slides™ Enumeration Indirect Stain Kit 2.0
User Manual

Introduction

Celselect Slides are used with the Genesis Cell Isolation System to capture and isolate individual
circulating tumor cells (CTCs) based on their size, utilizing microfluidics and 140,000 individual
microchambers. Isolated cells can be stained for immunofluorescence applications such as
enumeration and fluorescence in situ hybridization (FISH), or enriched for downstream analysis such ascell culture or single-cell genomics. After staining, the cells are counted on an automatic imager suchas the Agilent BioTek Lionheart LX Automated Microscope (recommended). The high capture efficiency
paired with excellent sensitivity and specificity make Celselect Slides the ideal approach to follow
tumor progression and response to therapy. The Genesis System and Celselect Slides technology
are used together to support users who want to optimize, modify, or create new protocols or custom
reagent formulations.
There are two approaches used to stain and identify CTCs. One utilizes indirect staining, where
primary antibodies attach to cell-specific surface antigens and are subsequently bound by secondary
antibodies that amplify the fluorescent signal. The second approach, direct staining, utilizes a
conjugated antibody that fluoresces specifically when bound to the antigen. Essentially, direct staining
achieves higher specificity at the cost of lower fluorescent signal intensity. Both methods permeabilize
the cell membrane, facilitating the use of additional cytosolic markers, as well as surface markers. This
user manual is for indirect staining. For direct staining, see the Celselect Slides Enumeration Direct
Stain Kit 2.0 User Manual (bulletin 3339).
Precautions■ 
Do not use reagents and consumables beyond the expiration
date on the label■■

Protect reagents from exposure to light sources■

Do not mix and match reagents from different lots/batches■

Fig. 1. The Genesis System.

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 isually inspect each blood sample for clots before processing;V
discard any clotted samples

 se protective barriers such as gloves, gowns, masks, and eye
Uprotection when working with whole blood specimens

Celselect Slides Enumeration Indirect Stain Kit 2.0 User Manual

List of Provided Components and Consumables
Table 1 lists components and consumables included in the
Celselect Slides Enumeration Indirect Stain Kit 2.0.

The waste jar (Figure 5) collects reagents and biohazardous waste.

Table 1. Celselect Slides Enumeration Indirect Stain Kit 2.0 (catalog #17009802).
Each kit supports processing of four samples.
Box 1 of 2. Shipment and Storage Conditions: Room Temperature
Consumables

Quantity

Celselect Slides 2.0

4

Reagents cartridge piercer

4

Waste jars and tubing

4

Inlet funnel 2.0

4

Priming reservoir

4

Fig. 5. Waste jar and tubing.

The Genesis System priming reservoir (Figure 6) is used during priming.

Box 2 of 2. Shipment and Storage Conditions: 4°C
Reagents

Quantity

Reagents cartridge

4

Celselect Slides and Consumables Box 1

Each of the Celselect Slides and consumables is intended forsingle use.
Celselect Slides (Figure 2) use Bio-Rad’s proprietary CTC
capture and analysis technology. Each slide has one laminatedside and the other side contains inlet and outlet ports. Opticalcharacteristics closely mimic a glass slide, rendering the slide fit
for imaging.

Fig. 6. Priming reservoir.

Celselect Slides Indirect Enumeration Reagents Box 2

The Genesis System dispenses reagents into the funnel in single
aliquots, and an entire set is provided for each run.
■Reagents cartridge — the reagents cartridge is shipped
preloaded with all necessary reagents to facilitate cell captureand enumeration

List of Equipment Provided with the Genesis System
Table 2. Equipment provided.

Fig. 2. Celselect Slides 2.0.

Blood and other reagents enter the Celselect Slides through theinlet funnel 2.0 (Figure 3).

Equipment

Catalog Number

Quantity

Celselect Slides Station 2.0

12020055

2

Reagents Cartridge Venter

CEL84093

1

Celselect Slides Imaging Adaptor 2.0

12019988

1

Wireless Barcode Scanner

CEL84092

1

Backflow Adaptor

12016521

2

The Celselect Slides Station 2.0 (Figure 7) is a single piece with aspring-loaded clamp to keep the Celselect Slides in place and incontact with the thermal vapor chamber beneath.
Fig. 3. Inlet funnel 2.0.

The reagents cartridge piercer (Figure 4) is firmly placed in the drip
plate of the Genesis System to penetrate the bottom of the reagent
cartridge and allow reagents to enter the inlet funnel.
Fig. 7. Celselect Slides Station 2.0.

Fig. 4. Reagents cartridge piercer.

© 2024 Bio-Rad Laboratories, Inc.

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Celselect Slides Enumeration Indirect Stain Kit 2.0 User Manual

Performing the Genesis System Self Test

The Celselect Slides Imaging Adaptor 2.0 (Figure 8) can hold up
to two Celselect Slides and is designed to fit into a standard PCR
plate format for use with an automated fluorescent microscope.

1.

Power on the Genesis System using the switch found on theback bottom left corner of the instrument (Figure 11).

Fig. 8. Celselect Slides Imaging Adaptor 2.0.
Fig. 11. Genesis System back panel.

The reagents cartridge venter (Figure 9) is designed to pierce the
top foil of the reagents cartridge for its use on the Genesis System.
Top-piercing is imperative to allow proper flow of reagents during
sample processing.

2.

Once the instrument is on, the Genesis System home screen(Figure 12) loads automatically and logs every run. From here,
the user can log in, gather system information, or shut down
the instrument.

Note: Log-in is not required.
Fig. 9. Reagents cartridge venter.

The backflow adaptor (Figure 10) is designed to connect theGenesis System vacuum pump to the priming reservoir, producing
positive pressure to prime slides.

Fig. 10. Backflow adaptor.

User-Supplied Equipment, Consumables, and ReagentsThe equipment, consumables, and reagents listed below arenecessary to perform CTC enumeration indirect staining with
Celselect Slides and can be purchased from any vendor.

Fig 12. Genesis System home screen.

Equipment Supplied by User

Single-channel pipets (20, 200, and 1,000 μl)
Serological pipet controller■ Test tube rocker■ Test tube rack
■ Automated fluorescence microscope■■

Consumables and Reagents Supplied by User

5 and 15 ml conical tubes
Sterile disposable serological pipets (5 and 10 ml)■ Phosphate buffered saline (PBS), 1x, pH 7.4■ Pipet tips (20, 200, and 1,000 μl)■ 70% ethanol solution■■© 2024 Bio-Rad Laboratories, Inc.

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Celselect Slides Enumeration Indirect Stain Kit 2.0 User Manual

3.

Place the waste jar in position at the base of the station bays
nearest the user (Figure 13).

7.

Press Start from the home screen to reach the Self Test
screen. Select each lane to initiate the Self Test (Figure 17).

Fig. 13. Place waste jar in position.

4.

Attach the waste jar tubing to the waste jar (Figure 14).
Fig. 17. Run Self Test.

8.

A confirmation list will appear on the screen, which lists theactions/items the system looks for prior to running the Self
Test (Figure 18).

Fig. 14 . Attach waste jar tubing.

5.

Press tubing into the pinch valve until you feel it click into place(Figure 15).

Fig. 18. Confirm Run Self Test.

9.
Fig. 15. Press tubing into the pinch valve.

6.

Ensure that the configuration is correct, then click Next toinitiate the Self Test protocol.

Attach Genesis System tubing to the waste jar outlet(Figure 16).

Fig. 16. Attach Genesis System tubing.

© 2024 Bio-Rad Laboratories, Inc.

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Celselect Slides Enumeration Indirect Stain Kit 2.0 User ManualPriming and Assembly
Note: While the slide is priming, the reagents cartridge can bepulled from 4°C storage and warmed at 37°C for a minimum of
10 minutes in an incubator or bead bath.

10. When the Self Test is complete, select 2.0 Kit Indirect
Enumeration from the Select Protocol options (Figure 19).

1.

Place the slide into the slide station 2.0 by gently pullingthe spring-loaded tab to allow the slide to fall into place.
The notched end of the slide drops into the notched endof the slide station (Figure 21).

Fig. 19. After a successful Self Test, the protocols can be selected.

11. Populate the data entry fields recording the reagents,
consumables, and slide identifiers using the barcode scanner(Figure 20).

Fig. 21. Inserting the slide into the slide holder.

2.

Place the slide station containing the slide on the
Genesis System. Firm pressure is required to snap the slide
station in place.
■■

The slit in the station prevents incorrect placement
 he slide station assembly should be pushed all the way
Tback until its vertical arms (finger holds) meet the clamping
tabs (stoppers) on the Genesis System lane (Figure 22)

Fig. 20. Populate data for run.

Note: If you are unable to scan barcode/QR codes, use thefollowing to manually enter the lot numbers with identifiers:
a. Celselect Slides Enumeration Indirect Stain Reagents (4-pack):“C” followed by the 6-digit lot number, e.g., C010101
b. Celselect Slides Enumeration Consumables (4-pack):“M” followed by the 6-digit lot number, e.g., M010101
c. Celselect Slides (4-pack): “S” followed by the 9-digit
serial number, e.g., S010101001

Fig. 22. Placing the slide station assembly into the Genesis System.

d. The Sample ID field is a free-form field allowing the user todefine a unique sample name for the run© 2024 Bio-Rad Laboratories, Inc.

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Project Title: CelselectVendor: Troutt Visual Services
File Name: Fig_10_CelselectDate: 03/01/24
Proof: 7 (final)

Celselect Slides Enumeration Indirect Stain Kit 2.0 User Manual

3.

7.

Slide the reagents cartridge piercer until it clicks or snaps into
place. Do this before placing the inlet funnel. The funnel is tootall to place the piercer after placing the funnel (Figure 23).

Pipet 8 ml 70% ethanol into the inlet funnel and start theenrichment run on the Genesis System.
Note: If 70% ethanol solution is prepared fresh, allow it todegas for at least 1 hour before use. Alternatively, the ethanol
solution can be purchased at 70% or made ahead of time.

8.

After priming, the instrument will prompt the user to remove
the backflow adaptor from the priming reservoir, discard
the ethanol, and replace the priming reservoir with the waste
jar tubing. Connect the luer of the waste jar tubing to the slide
completely by pressing down onto the outlet port. Twisting orover-tightening the luer may make it difficult to remove when
the run is complete.

Fig. 23. Bottom piercer hitting inlet funnel if not added in correct order.

Note: The ethanol will be in the priming reservoir after
completion.

4.

Tilt the inlet funnel under the piercer and attach it to the inlet
on the slide.

Best practice: To avoid introducing air into the slide, do notremove the ethanol that remains in the outlet port.

5.

Attach the backflow adaptor to the priming reservoir. Attach
the reservoir to the offset inlet on the right side of the slide.

9.

Be sure to attach the Genesis System tubing to the waste jar,and press the waste jar tubing into the pinch valve (Figure 25).

Note: The backflow adaptor can be difficult to attach to thepriming reservoir when dry. Lightly wetting the O-ring or theinside of the priming reservoir with a gloved finger or lint-freewipe saturated with either water or 70% ethanol can help
the pieces slide together more easily. Using buffer is notrecommended.
6.

Attach the Genesis System tubing to the backflow adaptor
tubing (Figure 24).

Fig. 25. Instrument loaded with slide, funnel, cartridge piercer, and waste
tubing properly routed through the pinch valve.

10. Pipet 8 ml 1x PBS into the funnel and press Resume on the
Genesis System screen to wash the slide.
11. The instrument will now prompt the user to dispense thesample and place the reagents cartridge.

Fig. 24. Genesis System tubing attached to the backflow adaptor in
preparation for slide priming.

© 2024 Bio-Rad Laboratories, Inc.

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Celselect Slides Enumeration Indirect Stain Kit 2.0 User Manual

Preparing the Reagents and Loading Reagents Cartridge
Note: The reagent cartridges must to be warmed at 37°C for 10 min
prior to use and left to sit at room temperature until use.

8.

Gently rotate the cartridge until it slides down the spindle,
taking care not to manually twist the spindle itself.

9.

Press Resume on the Genesis System screen.

1.

Make an appropriate dilution of the primary antibodies (1 ml isrequired per cartridge). For example, if making 1:200, then add
5 μl of the primary antibody to 1 ml of PBS. Add additional
primary antibodies to same solution.

Running the Genesis System
Below is a brief review of the steps for running your enumeration
sample after the slide has been primed.

2.

Make an appropriate dilution of the secondary antibodies(1 ml is required per cartridge). For example, if making 1:666,
then add 1.5 μl of the secondary antibody to 1 ml of PBS. Add
additional secondary antibodies to same solution.

1.

Add sample to the inlet funnel of each slide station (Figure 26).

Pierce the top foil of the reagents cartridge with the reagents
cartridge venter, making sure to twist the venter to open the foil.
3.

Add 1 ml of the primary antibody solution to chamber 7 ofeach reagents cartridge.

4.

Add 1 ml of the secondary antibody solution to chamber 9 ofeach reagents cartridge.

Fig. 26. Assembled slide station ready to have sample added to inlet funnel.

Note: Protect reagents cartridges from light and store at room
temperature until use.

2.

Sample Preparation and Processing
These steps can be done while the slide is priming. Slide priming
takes about 20 minutes to complete.
1.

If the biospecimen sample (blood) was kept chilled, allow thesample to acclimate to room temperature.

2.

Gently mix the biospecimen sample (blood) with a serologicalpipet several times. Draw up to 10 ml of sample per slide into
the pipet and dispense into a conical tube.

Pull aside the cartridge retainer arm and place the reagents
cartridge on the spindle, spinning it slowly until the keys alignand it drops down (Figure 27).

Note: The sample will be diluted, so the conical tube mustbe large enough to accommodate the final volume. For one
sample, a 15 ml conical tube would suffice.
3.

Dilute the biospecimen sample 1:1 with room-temperature
1x PBS. For example, 7.5 ml blood + 7.5 ml 1x PBS.

4.

Gently mix the sample by pipetting up and down several times
with a serological pipet.

5.

Before dispensing the diluted sample into the funnel, gently
mix the sample by either inverting the tube several times orusing a serological pipet.

6.

7.

Fig. 27. Reagents cartridge on spindle.

Gently dispense the sample into the funnel and avoid
generating bubbles. If large bubbles occur, they can beremoved with a pipet.
Important: Pierce the top of the reagent cartridge with thereagent cartridge venter before placing it onto the Genesis
System spindle.
Note: The cartridge has one lane labeled Start. This should
face the user.

© 2024 Bio-Rad Laboratories, Inc.

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3.

Release the cartridge retainer arm to its original position over
the reagents cartridge.

4.

Select OK on the User Action to initiate the rest of the run.

5.

After the run is complete, select Finish. Generally, sample
processing takes approximately 3.5 hours for 7.5 mlof sample.

6.

Remove the waste jar tubing from the slide.

7.

Remove the vacuum tubing from the waste jar.

8.

Dispose of the waste jar and tubing, the reagents cartridge,
the cartridge piercer, and the inlet funnel.

9.

Remove the slide from the slide station.

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Celselect Slides Enumeration Indirect Stain Kit 2.0 User Manual

10. Cover the ports of the slide with cellophane tape and be
sure to avoid wicking solution out of the slide (air decreases
imaging quality).

5.

Centrifuge the suspension for 3 min at 300 rcf or 4 min at
400 rcf. Discard media and replace with fresh prewarmed
media.

Note: When covering the ports with cellophane tape, avoid
covering the channels to limit imaging interference.

6.

Transfer 10 μl of cell suspension to a 1.5 ml centrifuge tube.

7.

Add 10 μl of Trypan Blue to the centrifuge tube and mix withcell suspension.

8.

Use 10 μl of the Trypan Blue/cell mixture to count cells ona hemocytometer.

9.

Prepare a dilution of 10,000 cells/ml in 1x PBS.

11. Store slides at 4°C until imaging. For best results, processslides as soon as possible.
Cleaning the Genesis System
Clean the Genesis System after each use by wiping the instrument
deck with either 10% bleach solution or 70% ethanol on a
towelette.

10. Add 50 μl of suspended cells to 4 ml of a whole blood sample.

Cleaning the Slide Stations
Clean the slide stations after each use by wiping them with either
10% bleach solution or 70% ethanol on a towelette.

11. Add 4 ml of PBS and gently mix by inverting the tube
several times.
12. Continue by returning to step 1 of the Running the Genesis
System section.

Disposal of Kit Consumables and Reagents
All used kit consumables and reagents should be treatedas biohazardous waste and disposed of in accordance withappropriate internal laboratory safety and environmental guidelines.

Table 4. Recommended Bio-Rad antibodies for spike-in studies.
Recommended
Dilution

Catalog
NumberPrimary antibody: anti-cytokeratin(pan reactive) (IgG1 isotype)

1:200

MCA6434GA

Primary antibody: anti-CD45 (IgG2a isotype)

1:200

MCA87GA

Secondary antibody: IgG1-FITC

1:100

STAR132F

Secondary antibody: IgG2a-Dylight650

1:100–1:500

HCA310D650

Antibody

Appendix
Specimen Collection, Transport, and Storage

Collect blood aseptically into EDTA tubes or Streck tubes(streck.com; see Table 3 for ordering information).
Caution: Once the tube is opened, process blood immediately.
Table 3. Specimen collection, transport, and storage.

Typical Staining Patterns If Using Cytokeratin, CD45, and DAPI

Streck Tube Order Information

Catalog Number

Table 5. Staining patterns.

Cell-Free DNA BCT RUO, 6-tube pack218961Cell-Free DNA BCT RUO, 100-tube box

218962Cell Type

Antibody Stains

CTCs

CK+, DAPI+, CD45–

Leukocytes

CK–, DAPI+, CD45+

Transportation
Temperature

Storage
Temperature

Shelf Life

Streck tubes

Ship at ambient
temperature;
do not put on ice

Store at room
temperature 18–30°C(65–86°F)

Process within
72 hours aftercollection

EDTA tubes

Ship on ice toRefrigerate sample
Process withinincrease theand bring to room
24 hours after
stability of the cells temperature, 15–30°C collection(59–86°F), before
processing

Tubes

Cytokeratin is a protein expressed in the epithelium and othercohesive cells. It is commonly recognized as a CTC marker and isgenerally not expressed on leukocytes. DAPI is a stain for nucleic
acids and is not expressed in anuclear cells such as erythrocytes.
CD45, otherwise known as protein tyrosine phosphatase or
leukocyte common antigen, is a surface antigen expressed bymost white blood cells. It is generally not expressed by cells
outside hematopoietic populations.

Cell Line Spike-In Guidelines

Below is an experiment designed to train the user on conducting
the enumeration protocol. It is a spike-in experiment using a
CTC line and blood.
1.

Wash a flask of MCF-7 cells with 1x PBS (example: 3 ml for a
T25 flask).

2.

Add 0.25% trypsin to the flask (example: 3 ml for a T25 flask)
and incubate at 37°C for 2–5 minutes or until cells detach.

3.

Add an equal volume of media to neutralize trypsin or otherdissociation reagent.

4.

Transfer cell suspension solution to a 15 ml conical tube.

© 2024 Bio-Rad Laboratories, Inc.

Antibodies are used in this panel to identify circulating cells
that may be sourced from a tumor with epithelial origins. These
antibodies may also be used to identify cells typically found incirculation to exclude them from analysis.
The open nature of the Genesis System allows these antibodies
to be substituted or complemented for different panels ofantibodies, assuming they have similar incubation settings and thatappropriate changes to downstream analysis are made.

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BIO-RAD and CELSELECT SLIDES are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions.
All trademarks used herein are the property of their respective owner.
© 2024 Bio-Rad Laboratories, Inc.

For Research Use Only. Not for use in diagnostic procedures.

Bio-RadLaboratories, Inc.
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