LOAD
CELLQUEST
Flowdata
> user screens, settings, and data >
SCREEN_displays > select a screen
display.
CONNECT
TO CYTOMETER
Acquire
> connect to cytometer. The Acquisition
Control Dialog Box with acquisition control functions:
Setup, Acquire, Restart, Save, Abort appears. The
setup box should have an X mark until you are
ready to start saving data. Move the window to the
side.
The
counter window is also under Acquire.
RETRIEVE
INSTRUMENT SETTINGS
Cytometer
> Instrument Settings > OPEN
> user screens, settings, and data >
settings > double click on a file >
SET (wait for 5 seconds,
while the world turns) >
DONE.
TO
SAVE DATA FILES
ASSIGN
DATAFILE NAME AND COUNT
Acquire
> Parameter Description > Folder
(Flowdata/user screens, settings, and data/FlowDataFiles)
> File (custom prefix = initials+YYMMDD,
and the file count = 1) > Enter sample
information in the comment box, if you will be using
ReproMan or ReproMac for analysis. Enter sample
information in the Sample ID box, if you will be using
CellQuest for analysis. Information can be entered in
both places.
ASSIGN
STORAGE INFORMATION
Acquire
> Acquisition and Storage >
Acquisition Gate (Accept all), Collect criteria (Event
count = # of cells to be counted, total or in a gate).
Storage gate (All or gated) If a gate is selected,
events outside the gate will NOT be saved for
future analysis. Resolution (256 or 1024) >
Parameters saved (turn off the parameters not in use) >
OK.
TO
CHECK SAMPLE SETTINGS
The
Setup box should have an X, while you run your
negative background etc. to adjust the high voltages
and compensations. Adjustments can be made either on
the cytometer or in CellQuest. To make the adjustments
in CellQuest, select Cytometer >
Detectors/Amps. Select Threshold and Compensation, if
these will be adjusted.
RUN
SAMPLES
Turn
the knob from Standby to Run. Swing the arm to the
side. Remove sheath tube. Put on your sample tube.
Swing the arm to the left. Pulse lights should appear
if cells are flowing. Select Acquire in the
acquisition control box. Make adjustments as
necessary. When putting the next tube on, there is no
need to switch to Standby. The sample probe is
continually flushing while in the run mode. When
satisfied with the settings, they must be saved, if
you want to use them again.
TO
SAVE NEW INSTRUMENT SETTINGS
Cytometer
> Instrument Settings > SAVE
> Find settings folder within user
screens, settings and data folder > type
in appropriate name > SAVE >
DONE.
TO
ACQUIRE DATA
Acquisition
Control Box > Pause > Abort >
remove the X from the Setup box. When the
correct sample is flowing, select Acquire. A tone will
sound when collection is finished and the file count
will increment. Change the comment or sample ID info
and run the next sample.
TO
QUIT CELLQUEST
File
> Quit > SAVE if you wish to
keep screen changes made during this run. The settings
are NOT saved as part of the screen. They must
be saved separately as explained above.
TRANSFER
DATA FILES
Copy
all your data to your directory on Ireland or on ZIP
disks. Do NOT save your data on the FACS
computer only. ALL files in the temporary
directories will be erased weekly on Monday morning.
PLEASE
RECORD ANY CELLQUEST OR INSTRUMENT PROBLEMS ON THE
CLIPBOARD LOCATED ON TOP OF THE FACSCALIBUR. THANK
YOU!
FURTHER
READING
There
is excellent FACS documentation at the website of the
Cell Analysis Facility of the Department of
Immunology: http://nucleus.immunol.washington.edu/Research_facilities/cell_analysis.html
For specifics on appropriate handling and waste procedures please see the
online chemical SOPs or our waste and spill notebook located in
room 352.
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