Quick Reference Guide to FACScan Operations
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TURN ON FACSCAN MACHINE FIRST THEN COMPUTER.
CHECK SHEATH RESERVOIR: (This should be done at the end of the previous user’s
session)
a. Open fluidics compartment. Optional to remove air filter, but do so gently.
b. Depress vent valve toggle switch.
c. Disconnect sheath’s white and blue tubing; and sheath probe cable.
d. Remove sheath reservoir; unscrew cap; and fill only ¾ to capacity with sheath fluid
(There is a fill mark)
e. Replace cap and reservoir.
f. Reconnect white and blue tubing; and sheath probe cable.
g. Ensure vent valve toggle switch is in up position and reservoir is pressurized.
EMPTY WASTE RESERVOIR: (This should be done at the end of the previous user’s session)
a. Disconnect waste’s white and orange tubing; and waste probe cable.
b. Remove waste reservoir; unscrew cap; and empty down drain.
c. Replace cap and reservoir.
d. Reconnect white and orange tubing; and waste probe cable.
e. Replace air filter gently by holding down all tubing and sliding filter into place.
CLEAR FLOW CELL OF TRAPPED AIR BUBBLES BY DRAINING AND FILLING:
a. Open the optics compartment door, above fluidics compartment.
b. While viewing the flow cell (quartz triangular cuvette on lower, right side), turn the
fluid control to "DRAIN" until you see that all the sheath fluid has drained from the
flow cell.
c. Next, turn the fluid control dial to "FILL" and watch as the flow cell steadily fills
with sheath fluid.
d. Repeat "DRAIN" and "FILL" 2-3 times, until no bubbles are visible upon filling.
e. Turn fluid control to "STANDBY".
NOTE: "BACKFLUSH" reverses the flow of sheath to flush fluid out of the sample
injection tube to help remove clogs. Ensure a tube of water and not your sample is
in the sample injection port at the time and follow "BACKFLUSH" with "DRAIN",
then "FILL" to refill the flow cell.
ENSURE FLUID CONTROL READS "HI" WHEN RUNNING WHOLE CELLS.
ON THE COMPUTER, LOG IN WITH USERNAME AND PASSWORD.
LAUNCH CELLQUEST PRO SOFTWARE FROM THE DOCK ICON.
UNDER THE "ACQUIRE" TOOL BAR,OPEN THE FOLLOWING FROM THE DROP DOWN MENU:
a. "CONNECT TO CYTOMETER": Automatically opens "ACQUISITION CONTROL
WINDOW"
b. "ACQUISITION": To identify what data is stored and how many events (cells) to
collect.
c. "PARAMETER": To identify where data is stored and how each sample can be
individually labeled.
i.
Click on "FOLDER" and locate your folder (or create your folder) ONLY in
the FACSCAN USER folder located on the Desktop or directly to your Zip
Disk.
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Click on "FILE", then "CUSTOM PREFIX", select "SAMPLE ID": This allows
you to enter a sample description or name in the SAMPLE ID block.
d. "COUNTER": To show the status of sample collection and number of collected
events.
UNDER THE "CYTOMETER" TOOL BAR, OPEN THE FOLLOWING FROM THE DROP DOWN
MENU:
a. "DETECTOR/AMPS": To select the parameters for your fluorochrome (discussed
later).
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Optional to open, "THRESHOLD": To establish the baseline level of detection.
Optional to open, "COMPENSATION": Used when two fluorochromes are used to
separate the overlaps in wavelength emission.
d. Optional to open, "STATUS": Provides status of laser, fluid levels and if at
"READY".
e. "INSTRUMENT SETTINGS": Allows you to save your optimized detector/amp
settings or to retrieve saved settings in your own folder/Zip disk, thus saving time.
UNDER THE "PLOT" TOOL BAR, OPEN THE FOLLOWING FROM THE DROP DOWN MENU TO
CREATE A:
a. "HISTOGRAM":
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"PLOT SOURCE", choose "ACQUISITION" to collect data or "ANALYSIS"
to view collected data files.
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"PARAMETER", choose the appropriate one for your fluorochrome.
b. "DOTPLOT":
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"PLOT SOURCE", choose "ACQUISITION" to collect data or "ANALYSIS"
to view collected data files.
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"X-PARAMETER", choose the appropriate one for your fluorochrome.
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"Y-PARAMETER", choose the appropriate one.
TO COLLECT DATA, LOAD SAMPLE:
a. Your are ready to collect data when the "READY" light on FACScan control panel is
lit.
b. Turn fluid control to "RUN"
c. Remember, use only FALCON polystyrene, 12 x 75mm tubes, #352054 which
fits properly on the sample injection port.
d. Turn the tube support arm to the right, and install sample tube on sample
injection port, then quickly center the tube support arm under the tube.
CLICK "ACQUIRE" IN THE ACQUISITION CONTROL WINDOW:
a. While the "SETUP" box is checked, you may view live data but the data will not be
saved. This setup mode is simply used to allow optimization of settings.
b. When you are finished with step 15, you should click the setup box (which removes
the "X") so that you can save sample data to files.
OPTIMIZE SETTINGS IN THE DETECTORS/AMPS WINDOW:
a. If you are acquiring sample for DNA analysis, click the "DDM" box then select the
DDM parameter for your fluorochrome.
b. Adjust the detector levels by moving the "VOLTAGE" to get the population on
scale.
c. Adjust the "GAIN" to allow you the choice of processing each of the signals
through a log or linear amplifier depending on your application.
d. In general, for common fluorochromes adjust the voltage for the parameters:
i.
SSC and FSC parameters: Used for FITC cell surface staining visualization.
ii.
FL1 parameter: Used for GFP visualization.
iii.
FL2 parameter: Used for Propidium Iodide staining for DNA cell
cycle/apoptosis analysis.
SHUTDOWN PROCEDURE:
a. Run FACS Clean in sample injection port for 5 minutes.
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Do not submerge black connection at top of sample port.
b. Run ddwater in sample injection port for 5 minutes.
c. Leave ddwater in sample port.
d. Turn to " STANDBY"
e. FILL SHEATH FLUID AND EMPTY WASTE AFTER YOUR SESSION
REGARDLESS OF HOW FEW SAMPLES YOU RAN.
DATA STORAGE:
a. Transfer all data to a removable storage media.
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Zip disk
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USB flash drive
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CD
b. Data should not be stored on the hard drive except temporarily if no storage media
is readily available; then remove from hard drive as soon as possible otherwise
inhibition of printer processing and system crashes will be experienced.
16. TURN OFF FACSCAN AND SHUTDOWN COMPUTER.
17. ASSURE ROOM DOOR IS CLOSED UPON EXITING.