How to use the Dunn School (TIG) FACScans

The TIG/Dunn School FACScans in Room 50

There are three Becton Dickinson flow cytometers available in Room 50 of the Dunn School - one FACSort (a newly upgraded 6 parameter, dual laser FACScan but with optional electromechanical sorting at up to 300 cells/second) and two standard FACScans (single laser, five parameter) that are permanently attached to FACSMate autosamplers (that allow automated aquisition to List Mode data files from either a 96 well plate or 24 individual tubes). Bookings are made on the form on the front of the RED FILE that also contains a log of all runs and copies of these instructions. Please note that all internal bookings/usage will be charged at 10 pounds per hour towards maintenance. At the moment we have a policy that non-departmental users can only use the machines through active collaboration with members of the Department.

The FACSMate autosampler

(A) How to use the FACScan and FACSmate autosamplers

NB. Main instructions are in BOLD FACE, with subsidiary information in normal type.

1) All samples must be fixed (2% formalin) for use on these machines - for live cells (or PI) use FACSsort. It is also important that samples in the tube rack or 96 well plate run contiguously, as the FACSMate assumes that it is blocked and throws an error if no cells are found in a well. Place either your tubes in the FACSMate tube rack from A01 (left rear) or your plate on the front plate holder (you must use Falcon 3910 plates only and ensure the plate is fully inserted on the holder with the well A01 at the left rear). Please ensure that you have at least 100 microlitres of each sample in 96 well plates, and at least 200 microlitres for tubes, which should contain a minimum of around 200,000 cells (even if you only want to collect data on 5000), in order to avoid errors due to the machine thinking it is clogged or has a loose connection!

2) Check waste bottles on FACScan (right hand bottle) and FACSmate (marked waste) are empty and fill the left hand SHEATH bottle on the FACScan and FACSmate (marked) with DI water (filtered if not from lab RO system) containing 0.1% sodium azide. NB. Make sure tubes are not kinked - they should face forward on the bottle caps. Check that the bleach (1% chloros in water) and water tubes are full and in positions D05 and D06 in the tube rack (unless you are running a full tube rack).

3) Turn on FACScan then FACSmate, followed by HP computer (check printer is ON, paper guide lever back, with green light on and enough paper), making sure the computer itself (bottom box) is switched on LAST.

4) Flip up switch between FACScan sheath and waste bottles to pressurize sheath bottle. [If bottle has leak or does not pressurize your samples will not run properly - get help from Steve/Sue]. Also check that the filter is not full of air (ie the last person to use it let the sheath run out!) - if so you can purge it through the stoppered tube in the top of the filter.

5) Switch fluid control from Standby to RUN for a few minutes. Then turn to DRAIN for at least 30 secs, back to FILL for 30 secs, and finally back to RUN . The fluid control should remain in the RUN position throughout unless you suspect a bubble in the flow cell (ask Steve how to check) or a blockage (ie your samples wont run or the scatter pattern looks odd - usually very little forward scatter when this happens).

6) The computer should eventually boot up the MASTER PAGE containing boxes to click on for the different programs - click the box marked FACSMATE (use left mouse button) to run the FACSmate control program (FCP).

7) Check the FCP main settings are for FACScan and FACSmate and select either 96 WELL PLATE or TUBE RACK as appropriate, and either 4 parameter 256 resolution (1 or 2 colour fluorescence) or 5 paramter 1024 resolution (3 colour analysis). Most changes can be made in FCP by using the cursor/arrow keys to move around the screen (easier than the mouse) and then hitting either the NEXT or PREVIOUS keys to see what options are available. Ocassionaly you will have to enter things like filenames or well numbers by typing from the keyboard. In any case, to activate any changes you make you must move to another field (with the arrow keys). Information on the machines status and other messages appear from time to time in the box at the bottom of most screens. Other options are available from the FUNCTION KEYS as depicted along the bottom of the screen (F1-F4, then a gap, and then F5-F8).

8) If this is the first run of the day you should perform a PURGE and DRAIN (P & D) by pressing function key F5 . If you get a message "Attempting to Initialise FACSMate: check connections between Computer and FACSMate" that does not go away after a few seconds you may need to switch the FACSMate Off and then On again and retry after the FACSMate has finished moving its syringes up and down (and beeped). If the problem persists you may need to then switch the computer Off and On to reboot it also (the computer itself - the bottom box - should generally be the last item to be switched On).

9) You can then select whether you wish to run by ROWS or COLUMNS by pressing F1 to DEFINE the TRAY setup and then moving to the field at the top right of screen and using NEXT or PREVIOUS to change the selection before returning to the original screen by pressing F8 (Main).

10) Press F3 (Aquire) to go to the main FCP aquisition page . Here you can set the volume of sample (eg. 40microlitre), the flow rate (eg. 26microlitre/min), a manual transport volume (40microlitre) and number of mixing cycles (3 for plate) or vortex mixing (ON for Tube Rack) using the cursor/next/previous keys as usual. Set events to 5000 or as required (max 50000).

11) You should normally select LIST MODE for storing your data, and save it on the hard disk (Unit #11) into your assigned directory (a number normally between 2 and 5 - the default is 5) and with FILES using your initials and the FACS experiment number (eg SC545). The file TAG normally starts at 1 for the first sample and increments automatically with each sample (giving, for example, actual filenames together with their directories that would go #11:/5/SC545001 then #11:/5/SC545002 ...)

12) Press F6 to go to the FACSCAN CONTROL PAGE to check or set up appropriate instrument settings. Again, you can make changes using the cursor keys and either NEXT/PREVIOUS or by typing in numbers. Any changes you make are fully interactive with the FACScan It is usually best to start by restoring some SAVED SETTINGS available by pressing F6 (Restore) . The FACScan log book should list setup files that you or other people have used that may be appropriate or a good starting point. Such setup files should all be stored in directory 1 (ie #11:/1/filename). Type the filename , press F3 (Continue) and wait for it to be read from disk, and then press F6 (Restore) to actually send the settings to the FACScan and return you to the control page where the new settings should be shown. The settings of PBL1CLSS are a good start for 1 colour analysis, but you will need to adjust the PMT1 (FSC) amplifier for your cells (eg. lymph-nodes, spleen set to 2.0; total PBL set to 1.5; blasts or cell lines set to 1.0 or less - should you need to set less than one you must set the detector to E-01 rather than E00 and an amplifier setting around 7.0 [meaning 7x10**-1] ). When the settings are correct press F8 (Quit) to return to the aquisition page.

13) If you are running 2 or 3 colour samples and need to adjust the compensation settings refer to the special note at then end.

14) If you wish to set a gate to avoid saving data from dead cells or debris, you can do this by pressing F4 (GATE DATA) . You can set multiple gates, each of which will act on the sample well you set it on and all subsequent wells until another gate applies. (This normally means you set one gate and it is therefore set on the sample in well A01). Press F1 (Continue) and the FACSmate then actually sucks up the volume you had set from well A01 and runs it through the machine to try and collect 5000 events. After running the sample (any excess will be lost to waste!), the dot plot showing on the screen during data collection will then be shown for you to draw the gate on (usually FSC against SSC, but you could change to FSC against FL1 for example to collect only data on FL1 positive cells in 3 colour analyses). To draw the gate press F5 (Set Gate) and use the cursor keys to move the cross-hairs, hit the RETURN key once to anchor a corner of the gate before moving to the next point, and finally hit the C key to close the shape around your cells. Generally, it is a good idea to set a much wider gate than necessary and perhaps collect more events for later analysis, especially if you are not experienced in identifying live and dead cells, or lymphocytes versus neutrophils etc by the FSC and SSC patterns. Likewise, if you have very few live cells or a poor cell preparation it is better to set a wide gate or even no gate to avoid the FACSMate throwing errors when it fails to find any cells in your gate - you can always experiment with gates during analysis of the saved list mode data. To return to the main aquisition page hit F1 (Continue).

15) Before running make a final check of the settings on the aquisition page (especially START and END wells) . If you are running your last set of samples you should also make sure that you have set the bleach and water tubes to D05 and D06 respectively (if you have more than one plate or set of tubes you can avoid the washes bypressing the space bar when the cursor is in these positions). Check that the wash is set for the LAST sample only (move cursor to this position and hold down the NEXT key until LAST appears).

16) Press F1 to run the samples. If you set a gate it will warn you of this and you should press F1 again to continue .

17) Before running samples, the computer will start to print - usually a blank page, so you can switch the printer off and immediately back on, and then wind back this page, to save paper ( make sure printer is all OK before leaving machine to run unattended because any printer error will cause the FACSMate to lock up!

18) The FACSmate will now run through your samples and show you the data as it collects it on the screen. You can change the parameters you see while they are running by using the cursor keys to move to the parameter shown and use previous or next to change (eg. you might want to see a dot plot of FL1 against FL2 and a histogram of FSC). You cannot change the format of the screen from one dot plot and one histogram, but all four (or five) parameters of data are stored to disk in list mode.

19) Cells should run through the machine with an event count (shown at top left of screen) between 100-2000 (optimally about 500 cells/second). You can change this rate during a run by pressing F1 (Injection Rate) and entering a new value (in the range 10-110 or use previous/next keys) while the sample is paused. Pressing F1 again continues sample running.

Please FILL IN THE FACSCAN LOG BOOK before you leave the machine unattended.

20) If you are running low on sheath fluid during a run you can use F1 (Injection Rate) to pause the current sample, and then release the pressure switch between the bottles on the FACSCAN to refill sheath and empty waste. After replacing bottles remember to flip up pressure switch, and it is a good idea to repeat the Drain and Fill procedure in step 5 above before hitting F1 to continue sample run.

21) If necessary (eg. you forgot to set something important like a gate or the machine settings!), you can abort the run (although only after the next sample has been aspirated and therefore lost!) by pressing F7 (Abort Tray).

22) If a FACSMate error ocurrs during a run it will stop and show a red error message in the box at the bottom of the screen and will usually ask you to acknowledge the message (often you need to acknowledge more than once). In most cases this will abort the run and the program will jump back to the first menu page. You can then attempt to restart the run by pressing F3 to go to the acquisition page, changing the START and END wells as appropriate (the printout should confirm where you had got to) and pressing F1 to start. The most likely error is one caused by a failure to find any cells in the well (check on microscope!). The same errors can occur if there are leaks anywhere (especially small FACSMate syringe seal), bubbles in the flow cell (check sheath fluid or Drain and Fill), the FACScan shows "Not Ready" status, you have fixed your cells to the plate by using Formalin without any BSA (resuspend vigourously) or ocassional more serious FACSMate faults (eg. it missed the well or tube completely or you used the wrong make of plate).

23) At the end of the run the machine will return to the FACSMate control program main menu page. Check on the printout that all your samples have been collected.

24) You can now switch off the FACScan and FACSMate machines at the front panel main switches. When the FACScan is off you should release the sheath pressure (push down switch between bottles) and then switch the fluid control to STANDBY. You should now transfer your data to the Macintosh Computer for analysis in CellQuest, and delete your files from the FACScan Computer Hard Disk before switching it off. Exit from the FACSMate Control Program by pressing F8 (quit) and confirm, to take you back to the MASTER PAGE.

(B) Transferring files to the Macintosh Computer for analysis in CellQuest

(assuming you know something about Macintosh "Basics")

1) Assuming the Hewlet Packard FACScan computer is still switched on and showing the MASTER PAGE, click the button marked FACSNET using the mouse.

2) Ignore the various messages that appear on the screen, especially DO NOT press return if it asks you to!

3) Eventually the FACSNET MAIN MENU page should appear, with options and function keys marked in a similar fashion to the FACSMate Control Program. Press F5 to go into SERVER mode. The screen should then blank but a message "PWS Server - Press Space to Break" should appear. Do not press the space bar at this time as this will take you back to the menu page. If the screen stays completely blank the program has crashed; to rescue press the STOP key (top left of keyboard) and as many return keys as needed to get back a menu (usually the FACSNET menu) and press F8 to exit FACSNET taking you back to the MASTER PAGE where you can start again from step (1).

4) When in SERVER mode you can access the files from any of the networked Macintosh Computers with CellQuest, FACSConvert, and Versaterm FTP Client installed (those in Room 50, 41 and 43). Note: if using the MOF drives, ensure that they are switched ON [green button glows] before you start the Macintosh or they may not be recognised even when you insert the disc.

5) Run Versaterm FTP Client from the Apple Menu, and attempt to Login to the appropriate FACScan (I or II) by clicking on the choices that appear. A login box should appear with you attempting to log in as the user FTP with password FTP and it is asking for your account. Hit the space bar (space is the account name) followed by return or clicking on the OK to complete the Login procedure .

6) After a brief wait the contents of the FACScan Computer's hard disk should appear, with the default folder set to #11:/5. Change the folder to your own by selecting the CHANGE DIRECTORY option on the drop-down menu obtained by click-dragging from the small triangle just above-right of the central window. Enter the full Hewlett Packard name of the folder you want to change to (eg. #11:/4), then click OK and the files should appear. Note that on some Macintoshes you need to press ALT rather than shift plus 3 to get #.

7) Select your files by clicking on the first in the list and then shift-click-drag down the list to highlight selected files.

8) Click on the RECEIVE button.

9) Choose a folder, or make a new one (usually on your MOF disc) and then click the SAVE button to actually transfer files.

10) When the files have all transferred (ie the selection has all been cleared) run FACSCONVERT from the Apple Menu.

11) Use the standard Macintosh Filer-type operations to find and select the files you have just transferred in the left hand window and to select a destination folder for the converted files. If you are converting the files back into the same folder you need to switch on the option to delete files after conversion (a cross appears in the small box when the option is on). Click in the CONVERT box to start the conversion , and confirm that you do want to delete the files afterwards. When the conversion is finished you can check that everything was OK by looking at the Conversion Log available from the File Menu at the top of the screen.

12) Quit from FACSConvert (don't save the Conversion Log) and your files are ready to use in CellQuest.

13) You should now delete your files from the FACScan Computer , most easily using the Versaterm FTP Client program as you should still be logged on. Select your files in the window and use the drop down menu available from the small triangle (as for changing the directory) but choose REMOVE. Confirm that you want to remove the files. The files should eventually disappear from the window and you can then click on the DONE button. You can now close Versaterm FTP Client by clicking in the window close box. You should also fill in the file details and the fact that you have deleted the FACScan Computer files in the LOGBOOK.

Using CellQuest for Analysis of Data

14) To use CellQuest it is often best to start with an old analysis document , some examples of which should be on the hard disk, such as "Standard 6 File Analysis", that can be loaded by double-clicking on the CellQuest Document.

15) You may get any error saying that CellQuest could not find the files - click OK and carry on anyway as you want to load your own files into the analysis document.

16) Click to highlight each dot-plot or histogram that you want to load your file into and either choose FORMAT DOT-PLOT or FORMAT HISTOGRAM from the PLOTS MENU at the top of the CellQuest Screen. FORMAT is the most important command in CellQuest. You can also use the KEY SHORTCUT obtained by holding the "Apple Key" (bottom left of keyboard) and pressing the F key. A box should appear allowing you to select a file and change various properties of the selected plot and when you are happy Click OK or press RETURN. You can adjust gates and markers etc. mostly by clicking and dragging them or by clicking and using copy and paste from the Edit Menu. If you wish to make totally new plots in your analysis document, this can be done by first clicking on the appropriate button in the palette (usually at the left hand side) and then clicking and dragging a box where you want the plot to be. When you have your analysis document the way you want it it is a good idea to click on Save As from the Files Menu and save it into the same folder as your data files (this allows you to reload/reprint etc without having to load all the files into the standard document again from scratch). The best way to learn about CellQuest is to play with some data in it!

17) When you are happy with the page you should check that the correct printer is selected in the CHOOSER (from the Apple Menu) and that the File Increment (CellQuest Plots Menu) is equal to the number of files in the document.

18) Click on the edge of one of the plots and when it shows a a box with four corner "ears", choose SELECT ALL from the Edit Menu (so all your plots should now have "ears"). You can now choose Print (File Menu or "Apple+P") and Next Data File (Plots Menu or "Apple+]") alternately to load in pages of files and print them out.

20) Finally, when all your files are printed out, you can save your own analysis document, quit from CellQuest, and buy Steve a pint of bitter.

Using the FACSort

This machine (it is the forerunner of, and similar to, what is now called the FACSCaliber) is most often used as a MANUAL FACScan (ie without the FACSMate autosampler) but can also be used to sort populations for further in vitro experiments. Because it uses mechanical sorting and the same FlowCell as a FACScan it has the advantages that there is no loss of sensitivity compared to sorters using streams in air and although the maximum sort rate is only 300 sorted (single population) events per second, the yields of sorted live cells can be as high as 90%.

For more details on sort rates and recoveries on the FACSort see here

The machine is run entirely from CellQuest running on a Macintosh computer, so until I give more details here, try reading the section above on the use of CellQuest but remember to FORMAT your dot plots and histograms for aquisition rather than analysing from files. Other quick points to note are that the FACSort must be switched on BEFORE the Mactintosh, and you must CONNECT to the cytometer (from the CellQuest Analysis Menu) which will then give you access to the Instrument Settings and related menus. The slight change in the sorting flow cell also means that it is advisable to allow the machine to run water for 10-15 minutes after the usual starting up procedure (drain/fill etc).

Sorting live cells - hints for maximising recovery

For analysis and general use of the FACSort you should use (filtered) deionised water plus 0.1% azide as sheath fluid the same as the FACScans, but if you want to sort live cells you need to change to PBS without azide. It is recommended that you run filtered 70% ethanol in water through the whole machine (ie. from the sheath fliud bottle) for about 30 mins, with a sort gate switched on (to flush out the sort lines), followed by sterile filtered PBS, if you want your sort to be sterile. Cells for live sorting should still be in PBS 1% BSA 0.1% NaN3 (NOT FIXED!) and kept at 4 deg C (a cut-out ice box that allows you to pack ice round the sample injector is available), as the azide will be washed out during passage through the machine. Sorted cells should be collected into 50ml Falcon tubes precoated with PBS + 1% BSA and containing 5ml of neat, 0.2 micron filtered FCS as the sorted cells are very dilute (eg. sort rate = 300/s and collection volume is always 9ml/min equivalent to only 2000 cells per ml!! or 100,000 cells per tube). Try to remove the sorted cells from the machine as soon as the 50ml tube is full and place them on ice. Spin the cells down in the cold (eg. 1500rpm for 15 mins in benchtop centifuge - slighlty faster and longer than usual to maximise yield) and resuspend in the smallest possible volume of medium ready for culture/transfer/etc. Under optimal conditions it is possible to get back up to 90% of the cells that the machine has counted as sorted, but recoveries around 50% are more realistic in practice. It is actually possible to check your sorted cells for purity simply by taking a 1 ml sample straight from the 50ml Falcon tube, although they will be analysed at only a few events per second. When you have finished sorting, please return the machine to water plus azide and flush out all PBS (including the sorting lines) It would also be helpful if you could run FACSRinse (provided) through the sample injector for 10 minutes or so, particularly if you have had any problems with clogging or unstable scatter which would indicate that your samples are making the Flow Cell dirty.

Finally, if you are using PI, Calcium Fluors or other dyes which might contaminate future samples please run 70% ethanol through the sample injector followed by water to clean it out.

The FACSort can now also analyse FOUR COLOURS: click here for info!

Setting up colour compensations

If you are running samples stained with more than one fluorochrome (eg both FITC and PE) then you will need to either use instrument settings that someone else has generated and are known to work with your reagents, or you need to set up your own colour compensations as described

CellQuest/Macintosh Crashes

There seems to be at least four ways to lock up the Macintosh and stop CellQuest communicating with the FACSort - they probably all relate to anything that might cause a delay in the Macs' response:
1) Having a queue of printouts in the PrintMonitor while trying to analyse or sort - this is made ten times worse if you have any other printing problems, such as running out of paper, using the wrong printer driver in the chooser, or using certain versions of LaserWriter 8 that have a bug in the automatic cassette selection.
2) Having slow or incorrect versions of drivers for CD-ROMS or MO-Discs installed. Similarly, CellQuest does not seem to like PC formatted MO-Discs, particularly for aquisition while sorting, so use Mac formatted disks.
3) Having the machine connected to the network via Netware - my guess is some of the Netware Inits seem to clash with CellQuest and cause it to lock up unpredictably.
4) When the machine is connected to Ethernet (even just for printing) it seems to be important to make sure the network is not reaching saturation - our machine has caused many fewer problems since the network was reconfigured to filter out irrelevant incoming packets, so making the Network much more responsive.

If you should have problems with the Macintosh locking up, try restarting with ALT+APPLE keys held down until it asks you to REBUILD THE DESKTOP (say yes). If you still have problems, try turning off the MO Drive and use the Hard Disk instead, and copy the files onto your MO Disk when you have finished (after a restart).

This page last updated by Steve Cobbold 24th February 1999.