FACSCalibure Flow Cytometry Hand outs

Administrator: Rokhand Arvan, Dr Dennis Kaspers lab

Phone # (617)525 0744, rarvan@rics.bwh.harvard.edu

On Line Calendar for sign up

Use the following on line calendar to book a time for using the FACS machine. Username
and password will be provided upon completion of your training.
http://os.med.harvard.edu/cgi-bin/microcalendars.cgi?Op=UserLogin

Start up
1- Turn on FACSCalibur cytometer (on the machine, Low flow rate light is green
& Standby light is orange)
2- Turn on FACStation computer (laser lamp will warm up (5 min), while you are
checking the fluidics containers)
Note: Always turn on the cytometer before turning on the computer when
acquiring data. This enables the computer to recognize that the cytometer is
connected. When analyzing data, it is not necessary to turn on the cytometer.
3- Open the Fluidics (slide drawer) to check if the Sheath tank contains
Flow/Sheath buffer or the Waste tank is empty.
4- To fill the sheath tank, flip the vent valve toggle to depressurize
5- Remove the sheath metal bracket, disconnect the sheath tank connection tubes and
take the sheath tank out.
6- Fill only 75% of the sheath tank. (Avoid filling the sheath reservoir to its
maximum capacity, because when it is pressurized, fluid may be forced into the
air supply tubing, preventing proper pressurization.)
7- Replace the sheath tank/metal bracket/Connecting tubes/detection probe
connector (No kink in tube lines)
8- To empty the waste tank, disconnect the waste tank connection tubes, remove the
waste tank and discard the waste fluid in the sink.
Caution: It is good practice to empty the waste reservoir when you fill the sheath
reservoir. This prevents the waste reservoir from overflowing.
Important Note: If your samples contain hazardous material discard the waste
fluid at the end of your experiment based on proper bio-safety protocols. Also,
wear appropriate safety gloves/lab coat when handling waste materials.
9- Add 400ml of bleach (10% final volume) to emptied waste tank and replace the
waste tank/Connecting tubes (No kink in tube lines) on its position.
10- Flip the vent valve toggle to pressurize.
11- Check the filter and sheath tube lines for bubbles (Remove bubbles by using the
roller clamp for filter or disconnecting tubes and pressing the tip of the valve
against the wall of a beaker.)
12- Enter your user ID and Password.
13- Proceed to the next step (QC).

Calibration or Quality Control (QC) Test

QC is a regular test of the machine performance which will be performed by the
administrator. In QC test the FACSComp program will automatically performs PMT
voltage test, Time Delay calibration, Compensation, and Sensitivity test.
Important Note: You dont need to perform the QC test unless the administrator asks
you to do so. The QC data are saved in the QC log book as a reference.
1- For QC test, prepare two tubes, named A and B. Add 1 ml and 3 ml of sheath
buffer to tube A and B, respectively. Then add one drop of each CaliBRITE beads
based on table 1. (A four color setup is recommended. If no APC bead is
available, perform three color setup using PerCP.)
Tube A = unlabeled + APC
Tube B = unlabeled + APC + other three labeled beads
Note: Keep the CaliBRITE beads in Fridge (4C). The bead suspensions are
stable in 4C for 1 hour.
2- Open FACSComp software, enter your name as operator, click on Accept
3- Check if the beads lot ID is correct (lot IDs are printed on the box containing the
beads).
4- Check that both Lyse/Wash (LW) and Summary Report boxes are marked, and
click OK
5- On the Cytometer set the Flow rate on HI, install tube A, and press the Run button
6- Click on the Start, the program will automatically check PMT voltage & Time
delay calibration.
7- Install tube B (mixed beads), and click on the Start, it will perform compensation
& sensitivity test
8- Remove beads, install DI water, and set the machine on Standby mode.
9- A copy of QC data files will be saved in FACSComp folder if you had checkmark
the summary report box.
10- Quit the program and it will print a copy of todays QC result
11- Fill the QC log book and insert the printed QC sheet in the log book.
Note: The QC can be done manually if the automatic QC does not end in 75
seconds, or the event rate is low. In this circumstance we may change numbers in
Detect/Amp and Target Value window. But this is not recommended for nonadmin users.
Abbreviations:
Lyse/Wash (LW) = Ab is washed away in typical immuno-phenotyping protocols
[in Lyse/No Wash (LNW) protocol antibody is not washed away),
HLA-B27 Calib = A clinical assay protocol,
Coated Bead assay (CBA) = An ELIZA type protocol.

Optimization
To adjust the Cytometer setting for your experiment, you need to perform an optimization
test before acquiring any data.
1- Open CellQuest Pro program, it will open an untitled file for you.
2- You can save this file as an optimization file in your folder or use the Standard
Acquisition Document (Std Acq Doc) which is available in the software.
3- Go to Acquire > Connect to cytometer
4- Create a dot plot (Go to Plot > Dot plot, or use the tool box to draw a plot)
5- In Inspector window for this plot, select the file type as Acquisition Analysis
(The axis in the plot will change to FSC/SSC as X/Y parameters).
6- Go to Cytometer > open Detector/Amp, Threshold, Compensation and Status
windows.
7- Install unlabeled cells (or any isotype control) on Sample Injecting Port (SIP) to
adjust FSC Amp/Gain and SSC voltage in Detector/Amp window.
8- On Cytometer press RUN and MED Flow rate buttons
9- Make sure the Set Up box in Acquisition window is marked, then click on
Acquire
10- Change FSC-Amp/Gain and SSC-voltage to put the population of interest on
scale (locate the whole visible population in the middle of the dot plot window)
11- Adjust FSC in threshold window to get rid of debris
12- Make a polygon around the population of interest, and take the regions name
outside of the polygon region (R1)
13- To save your sample, remove the tube and install DI water
14- Make more dot plots for FL1/FL2 as X/Y axis for two color staining of FITC
versus PE (or any other combination that is needed and suits your experiment)
15- Add Quadrant to the plots from tool box (use copy/paste option to add the same
Quadrant to other plots)
16- Select all plots except FSC/SSC plot.
17- Go to Inspector window for these plots (multiple objects), and choose Gate >
G1=R1 (only the G1=R1 data will be shown in these plots).
18- If desired change the regions color (go to Gate>Gate list>G1=R1, change the
color of the checkmark box).
19- Install unlabeled cells and click on Acquire
Note: use Pause & Restart in Acquisition window to refresh the data whenever
you make some changes.
20- Adjust FL1 and FL2 Voltage in Detector/Amp window to put the R1 population
(unlabeled) in LL quadrant of the FL1/FL2 plot
Note: After these adjustments you may close the Detector/Amp, and threshold
windows, because you wont make anymore changes in these settings.
21- Pause & remove the unlabeled tube.
22- To correct the spectral overlap between different detectors use the compensation
settings window. These settings are adjusted by using fluorescent positive cells.
Note: For each fluorophore, compensation should be set using the brightest
stained population. For example, in a panel containing CD3-FITC, CD4-FITC,

and CD8-FITC, set compensation using CD8-FITC, the brightest FITC-stained

population in the panel
23- Place FITC (+) control tube on the SIP, and restart Acquisition
24- Adjust FL2 - %FL1 in compensation window to place the FITC positive events
in LR quadrant of FL1 versus FL2 plot.
25- Remove the FITC tube, install PE (+) control tube on the SIP, and restart
Acquisition.
26- Adjust FL1 - %FL2 in compensation window to place the PE positive events in
UL quadrant of the FL1 versus FL2 plot (or adjust FL3 - %FL2 in compensation
window to place the PE positive events in LR quadrant of the FL2 versus FL3
plot).
27- Remove the PE tube, install PerCP (+) control tube on the SIP, and restart
Acquisition.
28- Adjust FL2- %FL3 in compensation window to place the PerCP positive events
in UL quadrant of the FL2 versus FL3 plot (it could be 0.0% change because
PerCP does not emit within the range of FL2 detector).
29- Adjust FL4- %FL3 in compensation window to place the PerCP positive events
in LR Quadrant of the FL3 versus FL4 plot.
30- Remove the PerCP tube, install APC (+) control tube on SIP, and restart
Acquisition.
31- Adjust FL3- %FL4 in compensation window to place the APC positive events in
UL Quadrant of the FL3 versus FL4 plot.
32- Close Compensation window.
Note: If you are using two colors, like FITC and PE, you dont need to perform
compensation for all the detectors. Only adjust the detectors relevant to your
experiment.
33- Pause and Abort in Acquisition window
34- Remove the tube and place DI water, go to Standby mode
35- Save this optimization setting in your folder (Opt-Doc).
(You may also save this setting in the Instrument Setting folder, Cytometer >
InstSetting)

Acquisition
At this stage, choose the data saving location and name, specify the data collection
information, and then, acquire data.
1- First create an Experiment Storage Folder for this experiment (you may use
todays date as its name). For example make a folder in: FACStation hard
disk/Home folder/FACS folder/Experiment storage folder (= todays date)
2- Continue with your optimization file that is still open.
3- Click on the Directory/Change button in Acquisition control window
4- Select the Experiment storage folder to save the data files in that folder
5- Click on the File/Change button in the Acquisition Control Window
6- Enter a file prefix name (the prefix can be the date you have performed the
experiment), and make sure the file count is set at #1(this refers to your tube #),
click OK, (the data file name will be: prefix + tube name)

Optional: You may set the Sample ID/Patient ID as the prefix, in that case you
have to enter each samples name in Sample ID box (in Acquisition Window)
every time you install a new tube.
7- Go to Acquire>Acquisition & storage, a dialog box will be open
8- Select the setting that fits your experiment, for example: collection criteria, # of
events=10000, Gate (G1=R1), Resolution=1024, and etc.
9- Click on OK to exit
10- Go to Acquire>Counters, open Counter window (Zoom on green button to make it
big)
11- Deselect Setup checkbox in Acquisition Control Window (this step is necessary
to save your data)
12- Do a final check (the right tube #, and so on)
13- Install tube #1 on the SIP, Press RUN, wait 5 to stabilize pressure
14- Click on Acquire
15- Live counting status is displayed, wait until you hear the Beep (your 10000 count
is done), remove tube 1, install tube 2 (Sample # 2), check if the data file # has
changed to #2, and click on Acquire
16- Continue the same procedure for all of your samples.
Note: If one of your samples does not reach the 10000 event counts, you may
pause and Save the incomplete data for that sample, and proceed to the next
sample. If you Pause and Abort, you have to read that sample again (or the order
of your tubes/data files will change).
17- At the end, place a fresh tube containing 1 ml of DI water on the SIP
Note: Please perform the clean up protocol (3 ml FACS Clean + 3 ml ddH2O
protocol mentioned in Shut Down section), if you are using viscous samples or
adhesive dyes such as, Propidium Iodide (PI); Acridine Orange (AO); Thiazole
Orange (TO). This will preserve a consistent function of the system.
18- Leave the cytometer on Standby mode, and depressurize the system if the next
user is arriving in one hour.
19- Otherwise proceed to shut down section. (For analysis you dont need the
cytometer).

Analyzing Data
1- Launch CellQuest Pro
2- Create a plot (dot plot), and draw a quadrant. You can use the plots in your
optimization file for your analysis (OR go to File > BD application>BD
CellQuest Pro>Sample file, and double click on Norm001 file)
3- Select a file in:
Inspector window > file > select data files. For example file #1>open (for FCS
Data Files you must create a plot first).
4- Select Stat > Quadrant stats, define statistical options in STAT window by using
Edit Quadrant Stat>OK
5- Save Data Analysis files as name-analysis file and place it in the same folder as
raw data files.
6- Always Log off from your account after you are done
5

Note: For exporting data you may use a Levy Jenning (LJ) data file format.

Shut down
1- Install a tube containing 3 ml of BD FACS Clean Solution (or 10% bleach) with
support arm to the side for 1 min (Vacuum ON). Set RUN on HI flow rate mode.
2- Move support arm under the tube for 5 min (Vacuum off)
3- Repeat steps 1 & 2 with tube containing 3 ml of DI water
4- Place a fresh tube containing 1 ml of DI water on the SIP
5- Go to Standby mode, and depressurize the system. Then turn off the Power button
on the Cytometer
6- Log off and Shut down the computer

Monthly Maintenance
Only administrator will perform this procedure.
1- Turn on the cytometer
2- Remove sheath tank and bypass the filter Disconnect the upper tubing of filter
from Saline filter port, connect the sheath tube (white) to the port labeled as
Saline Filter (Fig 1-4, pg 25)
3- Install a spare tank with 1-2 L bleach (10%) or BD FACS clean solution.
4- Install a tube with 3 ml of 10% bleach or BD FACS clean on SIP
5- Press RUN and High flow rate
6- RUN for 20-30 min
7- Remove tube from SIP
8- Repeat step 3-8 using:
a) BD FACS rinse solution,
b) DI water
9- Replace original sheath tank
10- Connect the sheath filter tubes
11- Place a tube with 1 ml of DI water on SIP
12- Press standby, depressurize and turn off the cytometer.
Note: If Cytometer doesnt work for three weeks, only use DI water for
maintenance to get rid of crystallization. Every 2 months, rinse air filter and dry
for 24 hours. Change sheer filter every 4-6 months.
Flurochrome
Excitation (nm)
Emission
Emitted color
Texas Red
488
615
Red
Phycoerythrin (PE)
488
575
yellow
Fluorescein
488
525
Green
isothyocianate (FITC)
PerCP
488
675
Orange
Allophycocyanin
595, 633, 635, 647
660
Orange
(APC)
For NERCE related inquiries please contact Christine Anderson, BSL-3 Animal and
Tissue Culture Core Laboratory, Rm. 632, christine_anderson@hms.harvard.edu