Caliburmanul 102006

Flow Core User Handbook FACSCalibur
FLOW CORE
USER
HANDBOOK
(OSX FACSCalibur)
3/16/2011
Table of Contents
• Training Information page 3

1. Pre training
2. Day of Training
3. Post training
• Policies page 4-5

1. Safety
2. Samples
3. Sign-up and Log
4. Cytometer
5. Computer and data storage
6. Acknowledgement
• Cytometer, computer workstation, and data management page 6-8

1. Cytometer
2. Computer workstation and printers
3. Software applications
4. Data management
• Start-up and clean-up procedures page 9

1. Start-up procedure
2. When you are finished procedure
• Acquisition and Analysis of data using CELLQuest Pro page 10-15

1. Acquisition of samples for phenotypic analysis
2. Acquisition of samples for cell cycle analysis
3. Analysis of data
o DNA page 16-18
• Four-color flow cytometry page 23
• Troubleshooting page 24
• WWW page 24
• References page 24
• Notes page 24
2
http://www.cancer.umn.edu/page/cores/flowcyte.html
TRAINING Information
Pre training
• Pre-training materials are available on the web and can be checked out from
Greg Veltri.
• Read “Introduction to Flow Cytometry”.
• Fill out the User information form.
• CD-ROM
• View CELLQuest Pro what’s new, acquisition, and analysis movies and
print tutorials
• Safety.
Users must provide documentation of Hazardous Chemical Waste Training to their lab safety
officer and to Sabina Fritz (Box 806), CCRB Lab Safety officer. The documentation must be
to Sabina Fritz within 30 days of security access activation. Without such documentation it
will be necessary to curtail activities and de-activate access to the CCRB labs until
documentation is provided.
Day of Training
• Bring the following items
• Complete User information form
• This handbook
• Introduction to Flow Cytometry, with questions completed
Post training
• Review CELLQuest Pro CD-ROM. Watch other software application
movies as necessary.
• Use handouts and flow charts as guides to operate the FACSCalibur and
CELLQuest Pro.
3/16/11 3
POLICIES
Safety
• Users must provide documentation of Hazardous Chemical Waste Training.
• Users must dispose of their sample tubes in their own laboratory.
• The core supplies you with gloves only and not lab jackets.
• No open toes shoes or shorts are allowed.
• In the event of a fire alarm, place the instrument in stand-by and leave the
premises. If not, you will be placed on probation or denied access by the core
manager.
• When disposing of the waste, a cover must be on the tank when moving to and
fro from the sink.
Samples
Cell populations to be analyzed on the FACS Calibur cannot contain radionuclides or
infectious agents. Specifically, it is not permissible to bring cells into the Flow Core
for analysis that contain radioactive substances (eg, P-32) or are known to be
infected with bacteria, viruses or fungi. Examples would include cells known to be
producing infectious virus such as EBV or HIV. This policy is enforced because flow
cytometers generate aerosols, which we cannot contain during analysis or sorting.
Sign-up and Log

• Sign up is done from our online web page
• If you are >15 min. late for your reserved time you forfeit that time if someone
else is waiting.
• You must cancel in advance of your reserved time. You are not required to use
all of your reserved time.
Cytometer
• Supply reservoir should be FULL and waste reservoir should be EMPTY.
• Leave no more than 1ml of H20 on SIP.
• Leave the cytometer in STANDBY if another user is scheduled after you.
• SHUTDOWN the cytometer and computer if you are the last user.
User Error: A clear example of operator error is when the air and vacuum lines on
the sheath and waste tanks are improperly connected. These lines are color
coded to allow for easy identification and connection. Improper connection of
these lines disrupts the fluidics of the FACSCalibur and requires a service call.
POLICY: Since this is a clear and preventable user error, the user will be placed
on probation and if it occurs again will banned from the core.
Computer and data storage

• ACQUIRE data files to the desktop, then TRANSFER data files to the server or
burn a CD.
• The core does not archive your data or templates, so please remember to do so.
• Connecting to the server/network or burning CD’s, please supplemental
handouts for that please.
Acknowledgement
• Please provide a reprint of your paper, so we can post it.
• Since our existence is based on our productivity please give the core staff
correct authorship and/or acknoweldgement.
3/16/11 4
3/16/11 5
CYTOMETER, COMPUTER WORKSTATION, AND DATA MANAGEMENT

Cytometer Anatomy
FACSCalibur
• Optics
• Laser and Photomultiplier tubes (PMT’s)
• 488 nm (FL1=515-545nm, FL2=564-606nm, FL3>670nm)
• 635nm (FL4=653-667nm)
• FL1 530/30 BP
• FL2 585/42 BP
• FL3 670 LP
• FL4 661/16 BP
• SSC 488/10 BP
• FSC diode 488/10
• Electronics
• Boards
• Fluidics
• Fluidics Drawer
• Sheath reservoir (left)

• Waste reservoir (right)
• Vent toggle switch
• Sheath filter
• Tubing and wiring
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• Sample Injection Port (SIP)
• Sample injection tube/Droplet containment system (DCS)
• Tube support arm Fluidics control panel

• LO (12 µl/sec), MED (35 µl/sec), HI (60 µl/sec) sample flow rate
• PRIME, STANDBY, RUN
• Options
• Loader
• Key pad
• FACSLoader and carousels (8)
• Collection station (sorting)
• Collection tubes
• Piesoelectric arm/Sort line
• Sort line purge buttons
Computer workstation and printer

• G3
• CD-ROM
• Zip drive
• Floppy drive
• Printer
• Storage Devices
• Hard drive (HD) for data acquisition
• 3.5” HD floppy disks
• CD-ROM Burner
• Network/Server
Software applications
• CELLQuest Pro
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• Multipurpose data acquisition and analysis software.

• CELLQuest (old)
• Multipurpose data acquisition and analysis software
• FACSComp
• Quality control for instrument
• Used for setup with 2nd laser
• FlowJo
• Multipurpose analysis application
• Loader Manager
• Controls FACSLoader. Used for performing instrument
clean-up, and in conjunction with Worklist Manager.
• ModFit LT
• DNA analysis software.
• Worklist Manager (WLM)
• Application for programmed acquisition of data. Used in
conjunction with Loader Manager and CQ Pro
Data management
1. CELLQuest Pro Files
• List mode data file or FCS files:
• Contains unprocessed data with all of the measured parameters from the
sample (raw data).
• Includes instrument settings.
• Experiment document or templates:

Contains all of the formats (plots, markers, statistics, etc.) when using
CELLQuest Pro. Data files are acquired and analyzed using experiment
documents.
• Instrument setting files:

Contains instrument settings, which can be saved and recalled. You can also
recall settings from a data file.
• Organization of files:
• Each Lab is given 100MB to store info on the Desktop, we suggest that you
create your folders as listed below:
• Suggested file structure
• Data files folder
• Experiment document/template folder
• Instrument settings folder
3/16/11 8
START-UP AND CLEAN-UP PROCEDURES
NOTE: If computer is ON and FACSCalibur is OFF, then shut down and turn off
computer, then perform start-up as above. This enables the computer to recognize that
the cytometer is connected.
1. Turn FACSCalibur ON (wait 30 seconds). Turn Loader keypad ON.
2. Turn computer ON. Cancel or connect to server.
3. CHECK waste (empty)/sheath (full).
4. PRESSURIZE the sheath and sheath filter lines. Flip toggle toward you.
5. PURGE sheath filter (remove air bubbles).
6. Set sample flow rate to HI. REMOVE dH20 tube.
7. Press PRIME (wait for instrument to go to standby), REPEAT.
8. When STANDBY button turns orange, then install dH20 tube and place place
support arm under the tube.
9. ACQUIRE samples after 5 minutes warm-up.
When you are finished

Cleaning Calibur
1. Place ~3ml 10% BLEACH solution in sample tube.

2. Install 10% bleach on FACSCalibur with support ARM TO THE SIDE for 1
MINUTE.
3. Put support ARM UNDER the tube and RUN on HI for 5 MINUTES.
4. Place ~3ml dH20 in another sample tube.
5. Install dH20 on FACSCalibur with support ARM TO THE SIDE for 1 MINUTE.
6. Put support ARM UNDER tube and RUN on HI for 5 MINUTES.
7.
8. Switch cytometer to STANDBY mode.
9. Leave <1ml dH20 on instrument.
10. FILL SHEATH reservoir with 3L of PBS (see mark on reservoir).
11. EMPTY WASTE reservoir, put ~400ml of bleach in reservoir.
12. Transfer your data files.
13. SHUT DOWN instrument and computer, and VENT the sheath tank if you are
the LAST USER.
3/16/11 9
ACQUISTION AND ANALYSIS OF DATA USING CELLQUEST PRO
Acquisition of samples for phenotypic analysis
The directions below are for acquisition of samples for immunophenotypic analysis.
A. CREATE or modify an acquisition experiment document.
1. Launch CELLQuest Pro From the Dock
(a new untitled experiment document is now open)
2. Choose Connect to Cytometer from the acquire menu. (The browser appears).
a. Resize to show only the acquisition control bar.
3/16/11 10
b. Move the browser to a convenient location.
3. Select the dot plot tool from the tool bar, click and drag on the experiment
document
4. Create another acquisition dot plot
3/16/11 11
5. Modify each plot so that you have FSC vs SSC and a FL1 vs FL2.
To modify a plot, click on an edge of the plot and choose plot type in the
inspector window.
B. SETUP for acquisition: optimize instrument settings and define regions.

1. Choose Detectors/Amps, Threshold, and Compensation from the Cytometer
menu and place them on the screen at a convenient location.
(In general the instrument settings menus (detectors/amps, threshold, and

compensation are dealt with in order, i.e. first detectors /amps are adjusted, then
threshold, then compensation). Remember compensation adjustments are
dependent upon the voltages applied to the different PMT’s.)
2. Choose Instrument Settings from the Cytometry menu.
3/16/11 12
3. Click Open, go to Desktop/ Lab Temp Instrument Setting folder/Calib file

and choose the Calib file.
4. Click Set and then Done on the Instrument Settings window.

5. Place Tube 1, subclass control (i.e. negative control), on the flow
cytometer, select RUN on the cytometer and click Acquire in the Browser
window.
6. Adjust the FSC amplifier, SSC detector in the detectors and amps window, so
that the bead population is located in the center of the plot.
The FSC and SSC amplifiers in general should be linear (exceptions for
example, are red blood cells or platelets).
Think of the forward scatter adjustment as focusing on a microscope.
You have a course adjustment (voltage: E00, multiplies FSC signal by 1;
E01, multiplies FSC signal by 10; E02, multiplies FSC signal by 100;
E03, multiplies FSC signal by 1000; E-1, multiplies FSC signal by 0.1)
and fine adjustment (amplifier 1.00-9.99). For side scatter and the
fluorescent parameters adjust the voltage.
The idea is to clearly identify your population on the screen. The more
you know about the relative size and complexity of your cells, the better.
7. Adjust the FSC Threshold to eliminate extraneous events, noise, or debris

by increasing the value to 120.
Thresholding is electronic gating. Events below a threshold value are
electronically excluded from acquisition. FSC is the usual threshold
parameter.
8. Define a region by selecting one of the region tools (rectangle, ellipse, or

polygon) on the tool palette.
9. Draw a region around the events of interest.
10. Select each plot and choose Multicolor Gating in the Inspector window.
11. Select the FL1 vs FL2 plot and choose Gate R1 from the pull down menu in the
Inspector window.
3/16/11 13
12. Adjust FL1 and FL2 detector voltages.
13. Place the population in the first log decade by adjusting the appropriate
fluorescent detector voltages. You may want to place a quadrant marker for
reference purposes.
14. Click Pause and Abort in the Browser window.
15. Remove Tube 1 from the cytometer and place Tube 2 (FITC positive
control) on.
16. Click Acquire in the Browser window.
3/16/11 14
17. Fluorescence Compensation the FITC.
PE (tube 2)
FITC (tube 1)
18. Once the FITC is compensated, repeat steps 15 thru 17 with Tube 3 (PE
control).
19. After samples are optimized, click Pause, then Abort in the Browser
window.
20. Place a tube of DI water on the SIP and place the instrument in Standby.
21. Close Detectors/Amps, Threshold, and Compensation windows.
C. ACQUIRE samples
1. Resize the browser to view the entire Browser window.
2. Define where to save the data by selecting Directory Change button.
3. Navigate to the Desktop and create a new folder. Once created, select this
folder.
4. Change the file name by first selecting on the File Button.
5. In the File window, change the prefix pull down menu to Sample ID
6. Close the File window and type in a sample name in the Sample ID text
box in the Browser window.
3/16/11 15
7. Type in labels in the Parameter Settings, so that you have labels on each
of your axes.
Label your parameters

Fill in the appropriate parameter label for each sample.
Example:
P1=FSC
P2=SSC
P3=FITC
P4=PE
P5=FL3
P6=FLA
P7=FLW or FL4
Time
8. Select Global Settings in the Browser window.
9. Click on Acquisition and Storage
10. View the inspector and observe the data storage changes that you could make if
needed.
3/16/11 16
3/16/11 17
11. Choose Counters from the Acquire menu and place on the screen. Expand the
Counters window to full size.
12. Place the Tube 4 on the cytometer in the RUN mode.
13. Uncheck the Setup box in the Acquisition Control bar of the browser.
14. Click Acquire (The setup box should be unchecked).
The sample will be acquired. After the beep, the data file is saved and the file number
will increment. Remove the tube and continue with subsequent tubes. Be sure to label
parameters or change sample description as needed.
D. When you are finished

1. Save your acquisition experiment document by selecting Save Document in the
File menu.
2. Navigate to your folder on the Desktop and save it there.
3. Save your instrument settings, by selecting Instrument Settings in the

Cytometer menu.
4. Select save and name your file, click done.
5. Complete the clean up procedure.
3/16/11 18
Acquisition of samples for cell cycle analysis
CREATE or modify an acquisition experiment document.

Launch CELLQuest Pro
From the Dock (a new untitled experiment document is now open) or open an existing
acquisition experiment document.
Choose Connect to Cytometer from the acquire menu.
The acquisition control bar appears and the cytometer menu is active. Move the acquisition
control bar to a convenient location. Note that the setup box is checked (if not check this
box).
Create or modify an acquisition dot plot with FL2-W and FL2-A as the x and y parameters
and no gate.
Select the dot plot tool from the tool bar, click and drag on the document to create a plot of
any size.
To modify a plot, select the plot and go to the plots menu and choose format plot, then
modify the plot.
Create an acquisition histogram plot for a single fluorescent parameter with FL2-A as the
parameter.
Go to the plots menu and choose your plot type and select the appropriate parameters. The
plot you create will be the same size as the first plot you created.
Place a marker (found on the tool palette) at channel 200 and another marker at channel 400
on the histogram plot.
Select Histogram Statistics from the Stats menu.
.
SETUP for acquisition: optimize instrument settings and define regions.

Choose Detectors/Amps, Threshold, and Compensation from the Cytometer menu and
place them on the screen at a convenient location.
In general the instrument settings menus (detectors/amps, threshold, and compensation are
dealt with in order, i.e. first detectors /amps are adjusted, then threshold, then
compensation). Remember compensation adjustments are dependent upon the voltages
applied to the different PMT’s.
Adjust instrument settings.
To restore settings, go to “instrument settings” and select saved instrument settings, click
set and click done. Instrument settings can also be retrieved from data files, simply select
the data file with the appropriate settings (while in “instrument settings”), then click, SET and
done.
Identification of cells/nuclei by DOUBLET DISCRIMINATION.
FL2-H should be in LINEAR mode and DDM (lower left corner of detectors and amps menu)
should be in FL2.
Adjust FL2-H until all of the events are on the FL2-W/FL2-A plot. Continue to adjust the FL2-
H such that the G0/G1 peak is at about channel 200.
Increase FL2-W until the single events are at channel 200 on the FL2-W parameter.
Threshold to eliminate extraneous events, noise, or debris.
Thresholding is electronic gating. Events below a threshold value are electronically excluded
from acquisition. Threshold on FL2 for cell cycle analysis.
Define a region.
Click on one of the region tools (rectangle, ellipse, or polygon) on the tool palette. Draw a
region around the singlets.
Format relevant plots with the defined region.
Select a plot, go to the plots menu and choose format. Turn the gate on in the plot source
menu.
3/16/11 19
Adjust background fluorescence (FL1, FL3, FL4) for any other parameters
Adjust the background fluorescence for each fluorescent parameter.
These parameters will be in Log mode
Fluorescence Compensation
Place the compensation controls on the cytometer and adjust the compensation for each.
OPTIONAL: Light scatter adjustments (FSC/SSC)
Analysis of light scatter is not required, but these parameters can be adjusted.
Place the sample on the flow cytometer and click Acquire.
Adjust the FSC amplifier, SSC detector in the detectors and amps window.
The FSC and SSC amplifiers in general should be linear (exceptions for example, are red
blood cells or platelets).
Think of the forward scatter adjustment as focusing on a microscope. You have a course
adjustment (voltage: E00, multiplies FSC signal by 1; E01, multiplies FSC signal by 10; E02,
multiplies FSC signal by 100; E03, multiplies FSC signal by 1000; E-1, multiplies FSC signal
by 0.1) and fine adjustment (amplifier 1.00-9.99). For side scatter and the fluorescent
parameters adjust the voltage.
The idea is to clearly identify your population on the screen. The more you know about the
relative size and complexity of your cells, the better.
Clearing the Screen
Click on Pause, then Resume or Restart in the Acquisition Control window.
After samples are optimized, click Pause, then Abort in the Acquisition window.
Close Detectors/Amps, Threshold, and Compensation windows.
ACQUIRE samples
As above.
When you are finished

As above.
ANALYZE data
Create the experiment document as described for aquisition of data, except the plot source
should be analysis, and you will select a data file to be analyzed
Consult the directions below for analysis of phenotypic data.
Analysis
• Analysis can be performed at the FACSCalibur or offline workstation. Other
applications like Attractors, ModFit LT, Paint-a-gate PRO, and FlowJo are available for
analysis.
• CREATE an analysis experiment document.
• Launch Quest pro
• From the apple menu (a new untitled experiment document is now open)
or open an existing acquisition experiment document.
• Create an analysis dot plot and include a data file and have FSC and SSC as
the x and y parameters and no gate.
• Select the dot plot tool from the tool bar, click and drag on the document
to create a plot of any size.
• Create an analysis histogram plot with a data file and the appropriate
fluorescent parameter (FL1, 2, 3, or 4) OR create an analysis dot plot or an
analysis density plot or an analysis contour plot for dual parameter acquisition
(e.g. FL1 vs FL2).
• Go to the plots menu and choose your plot type and select the
appropriate parameters. The plot you create will be the same size as the first
FSC/SSC plot you created.
3/16/11 20
• Add regions, gates and statistics

• Define a region.
2. Click on one of the region tools (rectangle, ellipse, or polygon) on the tool palette.
Draw a region around the events of interest.
3. Apply the gate (defined by the region) to the relevant .
4. Select a plot, go to the plots menu and choose format. Turn the gate on
in the plot source menu.
• Add statistics.
• Define histogram markers in a histogram plot using the marker tool from the tool
palette.
• Click and drag to define relevant boundaries.
• Add more than one marker.
• Define quadrants in a dual parameter plot using the quadrant marker tool from
the tool palette.
• Click and drag to define relevant boundaries.
• Choose histogram quadrant stats from the Stats menu.
• Print the file.
• Analyze the next or multiple files.
• To analyze single files one at a time:
• Deselect any selected plots, then choose Select All from the Edit menu.
• Choose change data file from the plots menu and select the next data file.
• Change any regions, gates, or statistics if needed.
• Print the file.
• To analyze multiple files simultaneously:
• Setup you experiment document to contain >1 data file.
• Choose File Increment from the Plots menu and change the file increment to
reflect the number of files to be analyzed simultaneoiusly.
• Deselect any selected plots, then choose Select All from the Edit menu.
• Choose Next Data File from the Plots menu.
• Print the file.
• Repeat the above sequence until all files are analyzed.
3/16/11 21
FOUR-COLOR FLOW AND USING THE RED DIODE (635nm) LASER

• Overview
5.The standard configuration for a FACSCalibur is a fixed 488nm argon laser. A
second 635nm red-diode laser has been added and can be used in conjunction with
the first laser for four-color acquisition of data.
6.You must optimize the instrument with biological samples for accurate acquisition
and interpretation of data. Controls for compensation are critical due to the number of
dyes being used.
• Instrument setup
7.Cytometer should be on and primed.
8.Perform Time-delay Calibration.
9.Prepare APC beads
10. Purchase APC beads from Becton Dickinson, cat#340487.
11. Add one drop of beads to 1ml filtered sheath fluid.
12. Open time-delay template from the apple menu and follow the directions in the
template.
13. Optimize instrument settings with biological samples.
3/16/11 22
TROUBLESHOOTING
14. Make sure sheath tank is pressurized.
15. Purge the sheath filter and prime the fluidics.
16. Run bleach and water.
17. Do not use contour plots in acquisition.
18. See Chapter 7 in FACSCalibur System User Guide.
19. See CELLQuest Pro User’s Guide.
WWW
• Flow Core web:
• http://www.cancer.umn.edu/page/cores/flowcyte.html
• Facts and information about the Flow Core
• Links to more sites
• Purdue:
• http://www.cyto.purdue.edu
• links to most flow sites
• Scripps
• http://facs.scripps.edu
REFERENCES
20. FACSCalibur System User’s Guide. Becton Dickinson Immunocytometry Systems,
1996.
21. FACS Training Manual. Becton Dickinson Immunocytometry Systems, 1995.
22. CELLQuest Pro User’s Guide. Becton Dickinson Immunocytometry Systems, 1994.
23. FACS Loader User Guide.
24. Becton Dickinson User Guides.
25. Current Protocols in Flow Cytometer. J. P. Robinson ed, John Wiley and sons, New
York, NY.
NOTES
3/16/11 23

Caliburmanul 102006

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Flow Core User Handbook FACSCalibur

• Training Information page 3

• Policies page 4-5

• Cytometer, computer workstation, and data management page 6-8

• Start-up and clean-up procedures page 9

• Acquisition and Analysis of data using CELLQuest Pro page 10-15

o DNA page 16-18

• Four-color flow cytometry page 23

• Complete User information form

• Introduction to Flow Cytometry, with questions completed

Sign-up and Log

Computer and data storage

CYTOMETER, COMPUTER WORKSTATION, AND DATA MANAGEMENT

• Sheath reservoir (left)

• Sample Injection Port (SIP)

• Sample injection tube/Droplet containment system (DCS)

• Tube support arm Fluidics control panel

Computer workstation and printer

• Multipurpose data acquisition and analysis software.

• Experiment document or templates:

• Instrument setting files:

START-UP AND CLEAN-UP PROCEDURES

When you are finished

1. Place ~3ml 10% BLEACH solution in sample tube.

ACQUISTION AND ANALYSIS OF DATA USING CELLQUEST PRO

Acquisition of samples for phenotypic analysis

1. Launch CELLQuest Pro From the Dock

(a new untitled experiment document is now open)

b. Move the browser to a convenient location.

4. Create another acquisition dot plot

B. SETUP for acquisition: optimize instrument settings and define regions.

(In general the instrument settings menus (detectors/amps, threshold, and

2. Choose Instrument Settings from the Cytometry menu.

3. Click Open, go to Desktop/ Lab Temp Instrument Setting folder/Calib file

4. Click Set and then Done on the Instrument Settings window.

7. Adjust the FSC Threshold to eliminate extraneous events, noise, or debris

8. Define a region by selecting one of the region tools (rectangle, ellipse, or

9. Draw a region around the events of interest.

12. Adjust FL1 and FL2 detector voltages.

14. Click Pause and Abort in the Browser window.

16. Click Acquire in the Browser window.

17. Fluorescence Compensation the FITC.

21. Close Detectors/Amps, Threshold, and Compensation windows.

1. Resize the browser to view the entire Browser window.

2. Define where to save the data by selecting Directory Change button.

4. Change the file name by first selecting on the File Button.

Label your parameters

8. Select Global Settings in the Browser window.

9. Click on Acquisition and Storage

12. Place the Tube 4 on the cytometer in the RUN mode.

14. Click Acquire (The setup box should be unchecked).

D. When you are finished

2. Navigate to your folder on the Desktop and save it there.

3. Save your instrument settings, by selecting Instrument Settings in the

4. Select save and name your file, click done.

5. Complete the clean up procedure.

Acquisition of samples for cell cycle analysis

CREATE or modify an acquisition experiment document.

SETUP for acquisition: optimize instrument settings and define regions.

When you are finished

• Add regions, gates and statistics

FOUR-COLOR FLOW AND USING THE RED DIODE (635nm) LASER