AlanFung:Protocol/FACScan

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BD FACScan (Becton Dickinson) User's Protocol[edit]

Shawn Meade, Alan Fung

Cytometer Appearance (Bottom Panel)[edit]

-------------   --------------  --------------
| Sheath    |   |.2um Sheath |  |   Waste    |
| Tank      |   |Filter      |  |   Tank     |
-------------   --------------  --------------

Cytometer Setup[edit]

  • Turn on the FACScan
  • Turn on the computer
  • Open both panels on the FACScan
  • Make sure the system is pressurized
(Vent valve should be in the upwards position)
(Sheath tank should be tight and stuck in place)
(No gap between the sheath tank and the top of the rack)

Purge bubbles from the sheath filter[edit]

  • Prepare a small waster beaker located near the sink
  • Pinch the tubing and remove white cap
  • Let drain into the small waste beaker until no bubbles are seen

Purge bubbles from flow cell[edit]

  • Open up the middle panel on the cytometer, locate the flow cell (a small window with fluid in it)
  • Move the side arm to the side
  • Remove tube containing MilliQ H2O form the sample injection port (SIP)
  • 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 SIP Line[edit]

  • Open the bottom panel
  • Locate the tubing linked to the orange valve leading to the waste tank (right)
  • Prepare test tube with MilliQ H2O
  • Open up the bottom panel
  • Reinstall the test tube with MilliQ H2O on the SIP and moving side arm back
  • Check wasteline (orange nozzle) for air bubbles (< 1 min)
  • Continue the filling function until tube is free of bubbles
  • Place cytometer on standby

CellQuest Setup[edit]

  • Open up 2 working pages (Side by Side)one for acq another one for analysis
  • Choose <Document Size> from the menu file
  • Click the square to the right of the highlighted square, and click <OK>
  • 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.

Analysis[edit]

Histogram Regions[edit]

  • Histogram regions are used to define subpopulations of data for analysis and to define gates, while histogram markers are used to generate statistics
  • Select the <Histogram Region> tool from the tool palette
  • Click to position the left edge of the region and drag to position the right edge of the region
  • Release the mouse button to complete the region

Deleting a Histogram Region[edit]

To Delete a region's definition

  • Choose Region List from the Gates menu
  • Select the region to be deleted from the Region List by clicking the selection arrows
  • Press <Delete>

Dot Plot[edit]

  • Choose Plot from the Plots menu
  • Set the X&Y parameter
  • Click the <Select File> button
  • Click <Open> to open the sample files foler
  • Click <Open> to open data file
  • Click <OK> at the Dot Plot dialog box

Setting a Gate[edit]

  • Click the Dash Region tool in the tool palette to select it
  • Position the cursor on the dot plot and click and drag to create a region

Applying the Gate[edit]

  • Choose Dot Plot from the Plots menu
  • Click Select File
  • Click <Open> to open data file
  • Choose FL1 for X parameter and FL2 for the Y parameter in the Parameter pop-up menus
  • Choose G1 from the Gate pop-up menu
  • Click <OK>

Load Sample[edit]

  • 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 on the FACScan after loading the sample

Complete software setup, acquisition and analysis[edit]

  • 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[edit]

Decontamination[edit]

  • Install tube (labeled with red tape) containing 2 mL of 10% bleach
  • Set the the <Running> mode on FACScan to <High Flow>
  • 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.

Sheath Tank Prep[edit]

  • Place paper towel on the panel
  • Depressurize sheath tank
  • Unscrew cap and remove tank, place cap onto paper towel
  • Top off to mark with BD FACSFlow buffer
  • Replace and repressurize.

Waste Tank Prep[edit]

  • 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
  • Replace waste tank
  • Pressurize the system and double check the system is close and tight
  • Close the bottom panel
  • Quit CellQuest
  • Turn off computer, monitor and cytometer

File Transfer to iMac for offline analysis (Optional)[edit]

  • 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