by Santosh Patnaik, July 2001

also see FACS tips, FACS - compensation , Fluorochrome features and FACS primer

Rm# 309, 3rd floor Chanin bldg.

Tel. 2724, Door keypad: 4-digit code, then *

1. Book time for using the machine. Appointment logs are available online at calendar.yahoo.com - username aecoms_scan_calibur and password is facs****. You have to log in to the computer. Also, you need a password/username to log in to the computer to use the machine - REMEMBER to log out at the end.

2. Have samples in capped Falcon# 2058 tubes and on ice.

3. There are two Becton Dickinson machines - FACS Scan and FACS Calibur. Calibur is the newer machine and has additional laser sources. Both machines use the same application program 'Cell Quest'. The application is used to acquire new data, i.e., run samples, and to analyze/plot data acquired previously. The application FlowJo is also available.

4. Open the machine to check levels in the sheath-fluid and waste-fluid chambers. Empty or refill as necessary. The sheath-fluid chamber is pressurized during runs; if the chamber is pressurized, you'll have to use the pressure-release switch before you can slide out the chamber.

5. The machine has to be powered on for it to be recognized by the computer. This recognition occurs when the computer is starting up. If the machine is powered off, turn it on and then restart the computer.

6. Put machine into STANDBY mode.

7. Launch Cell Quest application.

8. Setup a folder where your data will be stored. Acquire> Parameter description> Choose/setup new folder. As data files get collected, the application assigns them a name. The name is in the format 'Prefix.file-count'; e.g., XY122001.001. The file following it, into the folder, will be XY122001.002. You can set the prefix name in the same (parameter description) window. You can rename or delete files as you judge appropriate later on.

9. As each cell passes through the machine, it is measured for various parameters. The parameter P1 measures FSC, or forward scatter (~size of the cell). P2 measures SSC, or side scatter (~density/granulosity of the cell). P3: light emitted in FITC (FL1/green) channel. P4: in FL2/orange. P5: in FL3/red. And so on.

Again, use the 'parameter description' window to set P1 to FSC, P2 to SSC, and P3 or P4 or P5 or their combinations to FL1 or FL2 or FL3 depending on your experimental design. For example, if you are using a FITC conjugate, and are also using propidium iodide to gate dead cells, set P3 to FL1 and P5 to FL3.

10. Acquire> Connect to cytometer. This opens the 'acquisition control' window.

11. If you already know the instrument settings to use, remove the 'tick-mark' from 'set-up' in the 'acquisition control' window.

* Cytometer> Instrument settings> Open (a data file acquired previously using the instrument settings that you want to use again now)> Set> Done.

* If you don't have an old data file, but you know the numerical values for instrument settings: Cytometer> Detectors> Set to appropriate values.

* If you do not know the instrument settings to use, leave the 'tick-mark' on 'set-up' in the 'acquisition control' window. Then, File> New. Click on the 'dot plot' tool. A 'dot plot' window opens up: choose 'acquisition > analysis' in the pull-down menu and set the X- and Y-axes to desired parameters by choosing the parameter after clicking on the axis. Then, Cytometer> Detectors' and click on 'acquire' in the 'acquisition control' window. Have P1 and P2 set to linear scale and others to log scale. Increasing the values means increasing sensitivity/amplification. The dots (each representing one cell) will move further down the axes (higher values). Fiddle with the settings till you see that the cell populations obtain expected parameter values. Now remove the 'tick-mark' from 'set-up' in the 'acquisition control' window. Similarly, you can also do the setup with the histogram window (histogram plot tool). Fiddle with the detector settings till you see that the peaks are over the expected parameter values.

12. Acquire> Counter. This opens a counter window which shows the number and rate of cell-counts ('events').

13. Put machine in READY mode. If the display on the machine shows the machine is not ready, ensure that the waste and buffer containers are appropriately empty or full, and that the pressure switch is set to on. There may also be some other issue which can possibly be fixed by using the BACKFLUSH and DRAIN controls.

14. Briefly vortex your sample and put it in the sample holder under the steel probe.

15. Type the particulars about the sample in the 'parameter description' window and click on 'acquire' in the 'acquisition control' window. The counter will start running. Acquisition will stop after 10,000 events (you can change this value). You also have the option to pause, restart, or abort the acquisition. Once 10,000 events are up, the file will be saved automatically in the designated folder (see #8 above). If you want to stop earlier, pause> save.

16. Replace with new samples and repeat #15 above till all samples are done. Put machine to STANDBY mode before turning it off. Depressurize using the pressure release switch/valve inside the machine.

17. This can be done at anytime after data acquisition. You can use the Cell Quest application or you can use some other cytometry application such as Flo Jo.

18. In Cell Quest, File> New. Expand the window by dragging lower right corner.

19. Click on 'histogram tool' and drag the cursor to draw a rectangle of appropriate size. A new window will come up. Choose 'analysis' in the pull-down menu and 'select' the data file to plot. The Y-axis of the histogram, cell counts, can be set to appropriate scale here. You can also alter the drawing colors in this window. The X-axis of the histogram can be set to any of the parameters. Click on the X-axis on the histogram to open a pop-up menu and choose the parameter (e.g., FSC, FL1, etc.)

20. Stats> Histogram Stats. This will append another window with description of the file, e.g., sample particulars (#15 above) and acquisition date, and various statistical values of the data. You may want to remove some of the variables and values in the stats display to decrease the size of that window to fit it properly on the page. To do so, Stats> Edit Histogram Stats.

21. You can plot multiple histograms and save or/and print the page.

22. You can also overlay histograms to compare profiles. To do so, draw one histogram (#19 above) and then Plot> Overlay. A new window opens where you can choose the 2nd file and decide how it will be displayed (line pattern, color, etc.). After you have overlaid all the data files that you want to, Plot> Legend. This will append a small window showing which line corresponds to which data file. Print and/or save.

23. There are other ways to display data: 3D plots, contour plots, dot plots, etc. Familiarize yourself with the application to learn about them, and also to learn more advanced techniques like gating.