AlanFung:Protocol/FACScan
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BD FACScan (Becton Dickinson) User's Protocol
Cytometer Appearance (Bottom Panel)
------------- -------------- -------------- | Sheath | |.2um Sheath | | Waste | | Tank | |Filter | | Tank | ------------- -------------- --------------
Cytometer Setup
- Turn on both the cytometer and the computer
- Open up the bottom panel on the cytometer
- Make sure the system is pressurized
(Vent valve should be in the upwards position) (Sheath tank should be tight and non-movable) (No gap between the sheath tank and the top of the rack)
Purge bubbles trapped in the filter
- Prepare a small waster beaker located near the sink
- Squeeze the tube and pull white cap off from the tube coming out from the sheath filter
- Release the water with bubbles into the small waste beaker until no bubbles are seen
Removal of bubbles from flow cell
- Open up the middle panel on the cytometer, locate the flow cell (a small window with fluid in it)
- Swing the supporting arm to the side
- Take out tube containing MilliQ H2O
- Turn knob on the control panel to <Drain> in order to drain the flow cell (Notice the fluid level drops)
- Once the flow cell is drained, turn the knob to <Fill> (Notice the fluid level rises)
- Repeat the drain and fill procedure 3 times or until no bubbles are seen on the wall of the flow cell
- Leave the control knob on <Fill>
- Close the middle panel
Purge bubble from ? tube
- Prepare test tube with MilliQ H2O
- Open up the bottom panel
- Locate the tube linked to the orange valve
- Place the test tube with MilliQ H2O into the sample inlet
- Continue the filling function until tube is free of bubbles
CellQuest Setup
- Open up 2 working pages (Side by Side)one for acq another one for analysis
- Open template (saved on hard drive, for example: untitled/lo group/shawn/RCP-30-5-AA…)
- Or, create a new template and place plots.
- Set file name and path
- Under the Acquire menu, click “Parameter Description”
- create folder
- click on “file” button and set to “sample ID”
- click “OK”
- Enter name into sample ID line
- reset file count to zero
- close window
- Connect to cytometer (activates “Cytometer” on menu bar)
- Open up controls windows:
- &Detector amps
- Threshold
- Compensation
- Status
- Open instrument settings file
- Open an existing file under: Cytometer/Instrument Settings/…
- Or, the default: “untitled”/BD files/... Remember to resave it to your own folder.
Load Sample
- On FACScan set the <Fluid Control> dial to <Run> before putting in samples
- Using a transfer pipet, fill one of the plastic tubes with 2 mL of sample or more. Do not fill tube too high ~an inch high
- Turn side arm to side
- remove MilliQ tube
- Place sample on SIP
- Replace side arm
- Observe the pulse changes on the pulse meter ion the FACScan
Complete software setup, acquisition and analysis
- Set # of counts under: Acquire/Acquisition and Storage/…
- Optimize optical detector
- Set gate if necessary
- Acquire: Within the dialog box, uncheck setup and click acquire
- Save all templates and analysis work.
- Disconnect from Cytometer
- Close CellQuest
Shut Down FACSCAN
- Install tube (labeled with red tape) containing 2 mL of 10% bleach.
- Leave support arm to side for 1 minute on high flow
- Place support arm under the tube, make sure the cytometer is in run mode and let it run on high flow for 5 minutes.
- Repeat 1-3 with MilliQ water
- Leave MilliQ tube with 1 mL DI on SIP with support arm under tube.
- Depressurize sheath tank, remove and top off to mark with BD FACSFlow buffer. Replace and repressurize.
- Put cytometer in “Standby” mode
- Remove waste tank, empty into sink while tap is running. Refill with 100% bleach to a depth of about 1 cm.
- Turn off computer, monitor and cytometer
File Transfer to iMac for offline analysis (Optional)
- Disconnect from Cytometer
- Wake up iMac
- Go to “Chooser” on FACSCAN computer
- Select “Appleshare”
- Enter server IP “132.239.236.39”
- Enter name: “coreuser”
- Enter password: “bioeng”
- Connect as core user
- Close
- Double click “core user” folder on desktop
- Double click on “core user files”
- Create new experiment folder in your own primary folder
- Drag folder from “core user” to trash can once transfer is complete