AlanFung:Protocol/FACScan: Difference between revisions

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*Open an existing file under: Cytometer/Instrument Settings/…
*Open an existing file under: Cytometer/Instrument Settings/…
*Or, the default: “untitled”/BD files/...  Remember to resave it to your own folder.
*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.

Revision as of 16:12, 26 June 2009

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.