TITLE: BD Influx Cell Sorter Start Up and Shut Down for Viral Tagging and Grow
AUTHORS: Courtney Sanderson, Ho Bin Jang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The microbiome is now seen as the engine behind Earth’s nutrient and energy cycles, bioreactor and crop yields, and many diseases in humans, plants and animals. However, evidence is accumulating that viruses of these microbes modulate microbiome impacts. While virus discovery is occurring at a blistering pace, scalable experimental methods to link viruses to hosts, culture them, and phenotype them are needed. Here we improve understanding of a sequencing-enabled ‘Viral Tagging’ (VT) approach, and establish a variant ‘Viral Tag and Grow’ (VT+Grow) to rapidly capture and characterize viruses that infect a cultivated target bacterium, Pseudoalteromonas. First, benchmarking using model systems provided baseline cytometric and microscopy data for understanding how infection conditions and host physiology impact populations in VT flow cytograms. These advances are critical to interpreting, and increasing robustness across this challenging and finicky method. Next, we extensively evaluated and added an ‘and grow’ capability to evaluate where VT signals reflect adsorption alone or wholly successful infections that lead to lysis. Third, we applied VT+Grow to a clonal virus stock, which, coupled to traditional plaque assays, revealed significant variability in burst size – findings that hint at a viral ‘individuality’ parallel to the microbial phenotypic heterogeneity literature. Finally, we established a live protocol for public comment and improvement via protocols.io to maximally empower the research community. Together these efforts provide a robust foundation for VT researchers, and establish VT+Grow as a promising technology to scalably capture and characterize viruses from mixed community source samples that infect cultivable bacteria.</div></div>

[STEPS]
?. [Start up from Dry Shutdown]
After connecting to cytometer, go to file->workspace->QC Daily-> Courtney URFP file + click “restore laser delays”
?. [Start up from Dry Shutdown]
Place empty sheath tank on scale (with lid) and zero the scale and put the waste tank next to it
?. [Start up from Dry Shutdown]
Add 3L of MSM to sheath tank for a 1 day run
?. [Start up from Dry Shutdown]
Before connecting the lines, clean the connections with a wet kimwipe and MQ then connect all the lines
?. [Start up from Dry Shutdown]
Reconnect the fluid lines around the filter
?. [Start up from Dry Shutdown]
Rinse O-ring of waste tank before screwing the lid on (to help form a seal)
?. [Start up from Dry Shutdown]
Turn on vacuum then Jun Air then the air switch on the right side of FCM
?. [Start up from Dry Shutdown]
Add MQ to sonicator and add MQ to syringe + 0.2uM double filter
?. [Start up from Dry Shutdown]
Sonicate nozzle tip upright for 30 seconds then upside for ~15 seconds then right side up again for ~15 seconds
?. [Start up from Dry Shutdown]
Check stream by using the syringe on the nozzle tip and spraying against the edge of the sink
?. [Start up from Dry Shutdown]
Hold syringe upright and suck out excess water then VERY gently shake the rest of the excess out of the nozzle tip
?. [Start up from Dry Shutdown]
Without the nozzle tip, add the flush bucket to nozzle area then press rinse and let rinse for two seconds then run for two seconds
?. [Start up from Dry Shutdown]
Add nozzle tip
?. [Start up from Dry Shutdown]
Click rinse then take out filter from holder and tap it against the metal frame three times and replace the filter then run and backflush for 30 sec to see if sample line is clogged
?. [Start up from Dry Shutdown]
Bubble prime the nozzle:
?. [Start up from Dry Shutdown]
Turn on the lasers and make sure both lasers are at 100% power before proceeding
?. [Calibrating the Stream]
Remove flush bucket
?. [Calibrating the Stream]
Turn on illum button
?. [Calibrating the Stream]
While watching the stream pane, first adjust the black knobs above the nozzle to get the light centered above the waste drain then adjust the light intensity so it is slightly less than the most intense light possible
?. [Calibrating the Stream]
Watch pin hole and drop pane, use silver knobs (behind the black knobs just used) to finely adjust the stream
?. [Calibrating the Stream]
If the stream and pin hole can’t be aligned try sonicating the nozzle again and restarting at the bubble prime
?. [Calibrating the Stream]
Keep the back flush on and close shutters around nozzle, and press the open circle above where the shutter handle is to turn the laser on
?. [Laser calibration with Ultra Rainbow Bead]
Before applying rainbow bead sample, adjust sample pressure to +1 what the sheat pressure is at
?. [Laser calibration with Ultra Rainbow Bead]
Adjust the default display count to 200 and turn the red laser off with the blue laser on (the protruding silver poles on the left side of FCM)
?. [Laser calibration with Ultra Rainbow Bead]
Vortex ultra rainbow bead for 10 seconds
?. [Laser calibration with Ultra Rainbow Bead]
Backflush for thirty seconds then add the tube with ultra rainbow beads
?. [Laser calibration with Ultra Rainbow Bead]
Click acquire on computer then sample on FCM
?. [Laser calibration with Ultra Rainbow Bead]
Adjust the blue laser with the silver knobs with blue labels to the left of FCM. You want the laser to appear in the middle of the top pin hole
?. [Laser calibration with Ultra Rainbow Bead]
You want the value of the median around 45k and the RCV around 2
?. [Laser calibration with Ultra Rainbow Bead]
Turn the video knob next to the pinhole pane to video 2, adjust with right hand knobs to get an oval shape from the laser not a circle then place the laser over the top pinhole, this is the FSC adjustments and you’ll want a median around 25k for FSC with RCV around 2
?. [Laser calibration with Ultra Rainbow Bead]
Turn on the red laser, turn back to video 1, and adjust the red laser over the last pin hole with the red, silver knobs to the left of the machine, the value of the median around 45k and the RCV around 2.
?. [Drop Delay Calibration with Accudrop Bead:]
Switch the file in QC daily to “Courtney sorting” with laser delays checked
?. [Drop Delay Calibration with Accudrop Bead:]
Close deflection plates and turn on plate button to electrify plates
?. [Drop Delay Calibration with Accudrop Bead:]
Set to 1.0 Drop Pure
?. [Drop Delay Calibration with Accudrop Bead:]
Set drop frequency to 49.4
?. [Drop Delay Calibration with Accudrop Bead:]
Make sure the breakoff camera position is between 151-158
?. [Drop Delay Calibration with Accudrop Bead:]
Adjust the deflection angle to 67 degrees for both side streams
?. [Drop Delay Calibration with Accudrop Bead:]
Turn “test stream” on (not flash charge and short flash).Then, you can see two side streams (left/right) and mainstream (middle), respectively.  If two side streams from the mainstream are not bright enough, adjust first the black front facing knob to get all streams bright (if this is done, turn the lasers off and fine adjust the drop/pin hole panes again by adjusting silver knobs (behind the black knobs). Then, turn the test streams on again to check all the streams.
?. [Drop Delay Calibration with Accudrop Bead:]
Turn i) test streams ii)  flash charge and iii) short flash on and at each step, adjust the piezo amplitude (not frequency) to maximize side stream’s distance from the main stream.
?. [Drop Delay Calibration with Accudrop Bead:]
After adjusting streams, open the more tab and input the camera position number then +/- 20 to the other two variables. Then click set, this will adjust the drop delay number.
?. [Drop Delay Calibration with Accudrop Bead:]
Clean drain
?. [Drop Delay Calibration with Accudrop Bead:]
Set the default display count to 2000.
?. [Drop Delay Calibration with Accudrop Bead:]
Take out the accudrop bead from the fridge (you can see the precipitate on the bottom of the falcon tube) and vortex vigorously for 10 sec until the precipitate is completely gone. Run the drop delay bead. Click the acquire.
?. [Drop Delay Calibration with Accudrop Bead:]
Once you see the plot, adjust the daughter gates to what you expect will be 50/50 of the accudrop bead.
?. [Drop Delay Calibration with Accudrop Bead:]
Make a contour plot with SSC y-axis and FSC x-axis
?. [Drop Delay Calibration with Accudrop Bead:]
Click sort ready -> start then flip the knob with the orange tape above it from normal to the unlabeled orange tape (at this stage, you can see two side streams for P1 and P3, respectively).
?. [Drop Delay Calibration with Accudrop Bead:]
Remove bead and backflush for 30 seconds
?. [8 Peak Bead and Sorting Calibration]
After backflush, label a P1 and P3 tube and check the streams make any adjustments if the stream is being unstable
?. [8 Peak Bead and Sorting Calibration]
set default display count to 1000
?. [8 Peak Bead and Sorting Calibration]
Set the deflection angle to 45 degrees
?. [8 Peak Bead and Sorting Calibration]
Adjust P1 and P3 gates to where you expect the populations to be
?. [8 Peak Bead and Sorting Calibration]
Take out the 8 peak bead and vortex it for 5-10 sec.
?. [8 Peak Bead and Sorting Calibration]
Run the bead and adjust the gates, first adjust the parent gate in the contour plot (tightly gate the parent population with a 300% zoom) then adjust daughter gates: P1 and P3
?. [8 Peak Bead and Sorting Calibration]
Add tubes, sort ready-> start, then use a paper towel to check that the deflection angle is good and the sorted beads are going into the tubes
?. [8 Peak Bead and Sorting Calibration]
Run P1 and P3 and make sure the its % of parent is high, >95% from the parent is acceptable, want 97-100%.
?. [8 Peak Bead and Sorting Calibration]
Backflush for 30 seconds between samples
?. [Check 96 Well Tray alignment:]
Adjust streams if necessary (by adjusting the piezo value at each step of test stream, flash charge, and short flash),
?. [Check 96 Well Tray alignment:]
Change to 96 well tray sort and remove the 2 tube sorting apparatus then put the 96 well tray in
?. [Check 96 Well Tray alignment:]
Set to deflection angle of 70 degrees then test streams, flash charge, short flash to make sure the piezo amplitude is good
?. [Check 96 Well Tray alignment:]
Change deflection angle to 37 degrees (this will be flexible depending on how well the test stream is placed into the center of A1 well)
?. [Check 96 Well Tray alignment:]
In the tray control make sure you see 12 dots representing the 12 columns
?. [Check 96 Well Tray alignment:]
Sort ready
?. [Check 96 Well Tray alignment:]
Open the tray control pane. Click the A1 in the sorting pane (not tray control) to use A1 for the sorting test. Use the tray control to move the tray around to get the dot in the center on the directional pad press the center dot to have a test drop come out
?. [Check 96 Well Tray alignment:]
Click the blue circle in the tray control followed by home below the try control (make sure that the sort stream is deflected well from drain). Check the drop of A1 well. Adjust the deflection angle as well as left/right button in the tray control pane to get the drop to be centered on A1.
?. [Check 96 Well Tray alignment:]
Once the optimization of 96-well sorting is done, set up the FSC, SSC, SybrGold conditions for actual VTG.
?. [Check 96 Well Tray alignment:]
After you’re done with the sorting return to the 2 tube sorting option (click safe) then start shutdown
?. [Wet Shutdown - If you plan on using the machine the next day]
Stop running the sample then backflush for 30s
?. [Wet Shutdown - If you plan on using the machine the next day]
Turn off laser
?. [Wet Shutdown - If you plan on using the machine the next day]
Run a tube of MQ for 4 minutes
?. [Wet Shutdown - If you plan on using the machine the next day]
Turn up the sample pressure to around 23.4 so it rinses the line well
?. [Wet Shutdown - If you plan on using the machine the next day]
Leave the tube of MQ there but turn run off the sample button
?. [Wet Shutdown - If you plan on using the machine the next day]
Place the flush bucket under the nozzle and then the debubble reservoir
?. [Wet Shutdown - If you plan on using the machine the next day]
Fill the reservoir with MQ and make sure nozzle submerged
?. [Wet Shutdown - If you plan on using the machine the next day]
Stop the stream then purge for 1 minute to fill line with water
?. [Wet Shutdown - If you plan on using the machine the next day]
Stop the purge
?. [Wet Shutdown - If you plan on using the machine the next day]
Disconnect form cytometer
?. [Wet Shutdown - If you plan on using the machine the next day]
Shutdown cytometer
?. [Wet Shutdown - If you plan on using the machine the next day]
Close application
?. [Wet Shutdown - If you plan on using the machine the next day]
Shut down computer
?. [Wet Shutdown - If you plan on using the machine the next day]
Open plates and turn plate button off. Make sure stream is turned off.
?. [Wet Shutdown - If you plan on using the machine the next day]
Turn off auxiliary power
?. [Wet Shutdown - If you plan on using the machine the next day]
Remove fluid line on sheath tank
?. [Wet Shutdown - If you plan on using the machine the next day]
Set it on the FCM bench
?. [Wet Shutdown - If you plan on using the machine the next day]
Open pressure release on sheath tank
?. [Wet Shutdown - If you plan on using the machine the next day]
Wait till the pressure in sheath is 0
?. [Wet Shutdown - If you plan on using the machine the next day]
Turn air switch off
?. [Wet Shutdown - If you plan on using the machine the next day]
Turn main power off
?. [Wet Shutdown - If you plan on using the machine the next day]
Turn vacuum and jun air off
?. [Wet Shutdown - If you plan on using the machine the next day]
Remove lines from waste tank and empty waste tank, replace the waste tank but leave the lines disconnected
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Wash the sample line with 5% contrad for 1 minute. Sample PSI can be increased to around 23.5.
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Run/backflush for 30 seconds.
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Open the shutters in front of the nozzle
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Turn off laser
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Wash sample line with MQW for 4 minutes
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Occasionally (about once a month) place the flush bucket under the nozzle and then the debubble reservoir
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Stop backflush
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Stop the run
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Open the plates and turn the plate button off
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Put the flush bucket under the nozzle
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Depressurize the sheath tank and remove the sheath lines
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
If you think the waste tank is also already filled, you can empty the waste tank in this step and then reconnect the lines
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Dump the rest of the sheath tank then zero the tank again
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Add 1L MQ to the sheath tank and wash down sides w/ MQ, reattach the lines to the sheath tank, close the pressure valve and allow the repressurize
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Rinse and backflush for 1 minutes
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Run and backflush for 2 minutes
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Rinse and backflush until the scale reads 0.16kg then stop the backflush and rinse
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Depressurize sheath tank
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
DO NOT LET 5% CONTRAD INTO FILTER, disconnect the fluid lines around the filter and reconnect them to each other
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Add 1L of 5% contrad to the sheath tank
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Rinse and backflush for a few minutes
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Run and backflush for about 4 minutes to clean the sample line with a higher pressure
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Rinse and backflush until 5% contrad runs out
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Depressurize sheath tank
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Disconnect lines on waste tank then dump the waste tank
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Dump what is in the sheath tank as well
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Reconnect the waste tank and the sheath tank
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Zero the scale again (with lid) then add 4L of MQ
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Rinse of the connectors then reattach the fluid and air lines to the sheath tank
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
If there is a bubble in the nozzle, do a bubble prime to clean the nozzle
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Rinse and backflush till the tank runs out of MQ
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Switch to run and backflush as well
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Disconnect sheath and air lines from the sheath tanks and reconnect them to each other
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Take off the nozzle and put inside the machine
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Rinse and backflush for 5 minutes to dry the machine
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
After the dry time, disconnect the sheath tank to clean it by rinsing three times with MQ
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Clean the sonicator by rinsing with ethanol
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Stop the rinse and backflush
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Disconnect cytometer -> turn off cytometer -> shut down computer
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Turn off auxiliary power
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Remove waste tank and dumping the contents then rinse with MQ that is inside the tank
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Place both tank upside down next to the sink
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Turn air switch off
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Turn the vacuum off
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Turn the main power off
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Wash sample line with MQW for 5 minutes
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Stop backflush
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Stop the run
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Open the shutters in front of the nozzle
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Turn lasers off
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Open the plates and turn the plate button off
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Put the flush bucket under the nozzle
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Depressurize the sheath tank and remove the sheath lines
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Add 1L MQ water to the sheath tank and wash down sides with the MQ water, reattach the lines to the sheath tank, close the prssure valve and allow to repressurize
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Rinse and backflush for 1 minute and immediately stop the rinse and backflush
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Disconnect the blue connectors (above) from the filters and reconnect them to each other (caution: before disconnecting the blue ones, make sure that MQW completely stops. If not, when you disconnect the blue ones, MQWs spurts out of the lines)
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Do bubble prime for nozzle line with MQW by purge and pulse just for several times since if you repeat this many times then mqw in the sheath tank run out ( this bubble prime also uses the mqw in the sheath tank)
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Rinse and backflush until the sheath tank run out of MQW
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Stop rinse and backflush
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Disconnect the sheath and air lines (silver ones) from the sheath tanks and reconnect them to each other.
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Rinse/backflush to remove any remaining waters from the FCM lines (just for 5 sec?)
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Remove the nozzle tips and put inside the machine
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Rinse and backflush for 5 minutes to dry the machine
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
During the dry time, disconnect the sheath tank (open the depressurized valve on the sheath tank) to clean it by rinsing three times with MQ
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Stop the rinse and backflush
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Disconnect cytometer -> turn off cytometer -> shut down computer
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Turn off auxiliary power
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Remove waste tank rinse with MQ that is inside the tank then dump the rest
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Place both tank upside down next to the sink
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Turn air switch off
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Turn the vacuum off
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
Turn the main power off
?. [Start up from Dry Shutdown]
If the sheath tank runs dry during your run you have to recalibrate the machine with more sheath
?. [Start up from Dry Shutdown]
Wait for PSI of 10 in the waste tank
?. [Start up from Dry Shutdown]
Close the pressure valve on the sheath tank and turn the air switch on
?. [Start up from Dry Shutdown]
Wait for 20 PSI in sheath tank
?. [Start up from Dry Shutdown]
Add water to O ring of waste tank to help seal form (to be ~10 psi)
?. [Start up from Dry Shutdown]
If the psi of waste tank does not reach ~10 (e.g., ~5 psi), disassemble and reassemble the lines and lid
?. [Start up from Dry Shutdown]
If clogged, add falcon tube filled with MQ and start sample, if sample light won’t turn on then leave the tube for 1-2 minutes then remove the tube and back flush for 1-2 minutes; repeat process until sample light + backflush work as expected
?. [Start up from Dry Shutdown]
Leave backflush on during stream calibration
?. [Start up from Dry Shutdown]
If the machine has been shut down for over a month: add 3.5mL of 0.2uM filtered 70% ethanol to the debubble bucket then purge + pulse to clean the line, rinse the bucket 3x with MQ to clean it
?. [Start up from Dry Shutdown]
After the ethanol wash add debubble bucket with MQ then purge/pulse three times then remove the debubble bucket and allow the machine to run for 10-15 minutes before proceeding to actual bubble prime
?. [Start up from Dry Shutdown]
Put the debubble bucket under the nozzle and submerge the nozzle tip in MQ then purge till the line is full of MQ then pulse, repeat a few times
?. [Start up from Dry Shutdown]
If there are still bubbles appearing between the nozzle tip and the line then remove the bucket while purging then replace the bucket with more MQ
?. [Start up from Dry Shutdown]
Repeat this process till you get three purge/pulse cycles with no (or a very tiny) bubbles in the nozzle.
?. [Calibrating the Stream]
knobs can move the stream forward/backward (to adjust the brightness and focus) and left/right.
?. [Calibrating the Stream]
Remove any water from drain, make sure to not get kimwipe in the drain
?. [Calibrating the Stream]
In the pinhole pane, the stream should be sharply in focus and in line with the pin holes
?. [Calibrating the Stream]
Also you want the top of the nozzle to be slightly visible at the top of the pinhole pane, adjust this with the silver knob pointing straight up
?. [Calibrating the Stream]
In the drop pane, the stream should be close to the red tape marker
?. [Laser calibration with Ultra Rainbow Bead]
Sheath pressure should be 22, and the sample pressure around 22.9
?. [Laser calibration with Ultra Rainbow Bead]
Use the knob facing the front to adjust sample pressure and knob on the right side of FCM for sheath pressure if it is not 22 (but this will not happen often).
?. [Laser calibration with Ultra Rainbow Bead]
Prioritize making the readings of the beads cluster together in the plots by adjusting two knobs for the blue laser
?. [Laser calibration with Ultra Rainbow Bead]
Adjust the parameters if necessary (the parameters move the plots)
?. [Laser calibration with Ultra Rainbow Bead]
Also finely adjust once the beads are clustering and the numbers are close to what you want
?. [Laser calibration with Ultra Rainbow Bead]
Prioritize making the readings of the beads cluster together in the plots
?. [Laser calibration with Ultra Rainbow Bead]
Adjust the parameters if necessary
?. [Drop Delay Calibration with Accudrop Bead:]
When you change the frequency to 49.4, you can see that the shape of the mainstream is immediately changed (in the drop pane)
?. [Drop Delay Calibration with Accudrop Bead:]
By adjusting drop frequency (to roughly tune drop; ~49.4) and piezo (finely; 2.0-3.5), make the shape, position, & number (5.5) of drops as the figure below.
?. [Drop Delay Calibration with Accudrop Bead:]
Caution: If you could not get the shape, position, and number of drop like the figure (above) with the piezo (~2.0-3.5), drop frequency (~49.4), and camera position (151-158), you need to clean the FCM with 5% contrad followed by MQ. Please see the clean the FCM with 5% contrad below.
?. [Drop Delay Calibration with Accudrop Bead:]
If you cannot still see the bright side stream even though adjusting the black/silver knobs, turn off the plate on, open the deflection plate, clean the deflection plate with a wet kimwipe, remove MQ from plate with a dry kimwipe, and let it dry for 10-20min (do not close the plate). If this does not still work well, clean the plates again just with a dry kimwipe. Please put the picture you took here to see what is the best condition.
?. [Drop Delay Calibration with Accudrop Bead:]
Now decrease the drop delay to maximize the side streams and minimize the main streams
?. [Drop Delay Calibration with Accudrop Bead:]
Then stop -> safe-> stop in the software window->stop run the bead
?. [Drop Delay Calibration with Accudrop Bead:]
Remove paper towel under the two-tube sorting device with a new one and clean any water drops on the two tube sorting set up, which can cause the corrosion
?. [8 Peak Bead and Sorting Calibration]
Run for three minutes
?. [8 Peak Bead and Sorting Calibration]
Stop -> safe -> stop in the software window -> stop run the bead -> remove sample -> backflush -> add 150uL of MQ to each sorted population
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Use 0.02m-filtered 70% EtOH followed by washing with MQW three times. Specifically, put 0.02um-filtered 70% EtOH to reservoir and wash the nozzle line by purge and pulse and stop. Take out the reservoir and wash with MQW. Then, place the reservoir under the nozzle and put MQW followed by purge and pulse. Finally, if there is no small bubble in the nozzle, stop the purge and pulse.
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
disconnect the blue-colored connectors (plastic) that are connected to the filters (one is from the sheath line of the sheath tank to filter and the other is from the filter to FCM). Then connect the blues connectors to each other so that 5% contrad from sheath tank can directly go to FCM but not through the filter
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Use the waste in the tank (since it contains contrad) to rinse the sides of the tank at least three times
?. [Dry Shutown with 5% contrad wash - if you are shutting the machine down for 3+ days]
Double check that the two tube sorting set-up is in place
?. [Dry Shutown - if you are shutting the machine down for 3+ days and didn't work with sticky cells]
If you think the waste tank is also already filled, you can empty the waste tank in this step and then reconnect the lines