AlanFung:Protocol/FACScan: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
 
(16 intermediate revisions by the same user not shown)
Line 1: Line 1:
FACScan (Becton Dickinson)
==BD FACScan (Becton Dickinson) User's Protocol==
Shawn Meade, Alan Fung


==Cytometer Appearance (Bottom Panel)==
==Cytometer Appearance (Bottom Panel)==
Line 9: Line 10:


==Cytometer Setup==
==Cytometer Setup==
*Turn on both the cytometer and the computer
*Turn on the FACScan
*Open up the bottom panel on the cytometer
*Turn on the computer
*Open both panels on the FACScan
*Make sure the system is pressurized  
*Make sure the system is pressurized  
  (Vent valve should be in the upwards position)
  (Vent valve should be in the upwards position)
  (Sheath tank should be tight and non-movable)
  (Sheath tank should be tight and stuck in place)
  (No gap between the sheath tank and the top of the rack)
  (No gap between the sheath tank and the top of the rack)


===Purge bubbles trapped in the filter===
===Purge bubbles from the sheath filter===
*Prepare a small waster beaker located near the sink
*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
*Pinch the tubing and remove white cap  
*Release the water with bubbles into the small waste beaker until no bubbles are seen  
*Let drain into the small waste beaker until no bubbles are seen  


===Removal of bubbles from flow cell===
===Purge bubbles from flow cell===
*Open up the middle panel on the cytometer, locate the flow cell (a small window with fluid in it)
*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  
*Move the side arm to the side
*Take out tube containing MilliQ H2O  
*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)
*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)
*Once the flow cell is drained, turn the knob to <Fill> (Notice the fluid level rises)
Line 31: Line 33:
*Close the middle panel
*Close the middle panel


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


==CellQuest Setup==
==CellQuest Setup==
*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…)
*Open template (saved on hard drive, for example:  untitled/lo group/shawn/RCP-30-5-AA…)
*Or, create a new template and place plots.
*Or, create a new template and place plots.
Line 58: Line 67:
*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.
==Analysis==
===Histogram Regions===
*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===
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===
*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==
*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===
*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==
==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 the <Fluid Control> dial to <Run> before putting in samples
On FACSCAN set control dial to run
*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
*Turn side arm to side
remove MilliQ tube
*remove MilliQ tube
place sample on SIP
*Place sample on SIP
replace side arm.
*Replace side arm
Complete software setup, acquisition and analysis
*Observe the pulse changes on the pulse meter on the FACScan after loading the sample
Set # of counts under:  Acquire/Acquisition and Storage/…
 
Optimize optical detector
==Complete software setup, acquisition and analysis==
Set gate if necessary
*Set # of counts under:  Acquire/Acquisition and Storage/…
Acquire: Within the dialog box, uncheck setup and click acquire
*Optimize optical detector
Save all templates and analysis work.
*Set gate if necessary
Disconnect from Cytometer
*Acquire: Within the dialog box, uncheck setup and click <Acquire>
Close CellQuest
*Save all templates and analysis work.
Shut Down FACSCAN
*Disconnect from Cytometer
Install tube (labeled with red tape) containing 2 mL of 10% bleach.
*Close CellQuest
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.
==Shut Down FACSCAN==
Repeat 1-3 with MilliQ water  
===Decontamination===
Leave MilliQ tube with 1 mL DI on SIP with support arm under tube.
*Install tube (labeled with red tape) containing 2 mL of 10% bleach
Depressurize sheath tank, remove and top off to mark with BD FACSFlow buffer. Replace and repressurize.
*Set the the <Running> mode on FACScan to <High Flow>
Put cytometer in “Standby” mode
*Leave support arm to side for 1 minute on high flow
Remove waste tank, empty into sink while tap is runningRefill with 100% bleach to a depth of about 1 cm.
*Place support arm under the tube, make sure the cytometer is in run mode and let it run on high flow for 5 minutes.
Turn off computer, monitor and cytometer
*Repeat 1-3 with MilliQ water  
File Transfer to iMac for offline analysis (Optional)
*Leave MilliQ tube with 1 mL DI on SIP with support arm under tube.
Disconnect from Cytometer
 
Wake up iMac
===Sheath Tank Prep===
Go to “Chooser” on FACSCAN computer
*Place paper towel on the panel
Select “Appleshare”
*Depressurize sheath tank
Enter server IP “132.239.236.39”
*Unscrew cap and remove tank, place cap onto paper towel
Enter name: “coreuser”
*Top off to mark with BD FACSFlow buffer
Enter password: “bioeng”
*Replace and repressurize.
Connect as core user
 
Close
===Waste Tank Prep===
Double click “core user” folder on desktop
*Put cytometer in “Standby” mode
Double click on “core user files”
*Remove waste tank, empty into sink while tap is running
Create new experiment folder in your own primary folder
*Refill with 100% bleach to a depth of about 1 cm
Drag folder from “core user” to trash can once transfer is complete.
*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)==
*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

Latest revision as of 21:42, 9 July 2009

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