AlanFung:Protocol/FACScan

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Revision as of 16:13, 26 June 2009 by >Alan6017518 (→‎Load Sample)
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FACScan (Becton Dickinson)

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 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

  • Using a transfer pipet, fill one of the plastic tubes with 2 mL of sample or more. Do not fill tube too high.
  • On FACSCAN set control dial to run
  • Turn side arm to side
  • remove MilliQ tube
  • place sample on SIP
  • replace side arm.

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