FACSCalibur Training

General Information

The FACSCalibur is a useful analysis

tool.
The instrument has 2 lasers- 488
(primary) and 633 (secondary) that
can detect FSC, SSC, FL-1 (FITC)
FL-2 (PE) FL-3 (CYC, Tri-Color) and
FL-4 (APC).
Immuno-phenotyping (CD4, CD8),
Apoptosis (PI and Annexin), Cell
Cycle, and GFP analysis are just some
of the protocols that can be
established with the FACSCalibur.
It is very easy to use.

FACSCalibur Training
Appointments

All Flow appointments are scheduled through the Flow Facility Manager
Please arrive on time- very important
If you are scheduled for 1pm and do not show until 1:30, the investigator
will be billed starting at 1pm.
You are billed for the time that you are on the instrument in 15 minute
increments
If you arrive late and there is another appointment after you, you will be
asked to leave the instrument and resume at a later time.
Cancellations require at least 24hrs notice. Cancellations without proper
notice will be billed accordingly. Some exceptions will be made.
No appointments will be taken by email. Please call x3402 or come to
HEB 253 to schedule appointments.

FACSCalibur Training
General Flow stuff (Instrument)

If there is an issue with the instrument, do not try and fix it. If there is a
problem I will check the situation out and take the appropriate actions.
If the instrument is out of sheath fluid or the waste needs emptying please
contact me.
If you are using on the weekend and the sheath fluid is empty or the waste
is full you will have to take the appropriate actions.
Do not try to fix instrument under any circumstances
I will have the instrument ready for you to use during the weekdays.
If you are running on the weekend, you will have to set it up.

What is Flow Cytometry?

FACSCalibur Training
Flow Cytometry

Cytometry-The characterization and measurement of cells and

cellular constituents
Flow-To issue or move in a stream
Flow Cytometry, then, can be defined as the characterization
and measurement of cells and cellular constituents as they
travel in a fluid stream

FACSCalibur Training
Scattered and Emitted Light

Flow Cytometry deals with scattered and emitted light

Scattered light in the form of Forward scattered light FSC (Cell Size) and
Side scattered light SSC (Cell complexity/Granularity)
There are forward and side scatter detectors that detect the light and photomultiplier tubes amplify the signal and the computer turns it into a working
signal.
FSC and SSC are the 2 most important parameters

FACSCalibur Training
Scattered Light

488nm Laser

Cell Complexity

Cell Size

FACSCalibur Training
Scattered and Emitted Light

FACSCalibur Training
Emitted Light

Fluorochromes and fluorescent dyes etc emit light

due to their fluorescent properties.
Fluorochromes emit light in certain spectra not a
distinct wavelength
These spectra can overlap with one another causing
one color to be detected in another spectrum/channel
Spectrum overlap causes a need for compensation
which reduces this overlap
FITC, PE, PE-CY5 and APC are just a few of the
fluorescent stains that can be used.

FACSCalibur Training
Fluorescence

CD3 is a T-cell marker

CD19 is a B-cell marker

FACSCalibur Training
Laser Point of Interrogation and Hydrodynamic Focusing

Laser Point of Interrogation

488nm and 633nm lasers

1. Within the flow cell, a slow-moving sample stream is injected into a
faster moving "sheath" stream
2. Surface tension and laminar flow causes the sample stream to be
"wicked off" the injection point into a narrow, faster moving stream
within the sheath stream (stream within a stream).
3. Careful control of the velocity of the two streams allow for fine control
of the width of the center stream and, therefore, the alignment of the
cells within the center stream.

FACSCalibur Training
Light Scatter Parameters

Parameter
P1
P2

Detector
FSC
SSC

Forward Scatter
Side Scatter

Both are 488 excitable

The 2 most important parameters

FACSCalibur Training
Fluorescence Parameters

Parameter

Detector

Excitation

Emission

P3

FL-1 FITC

488

518

P4

FL-2 PE

488

575

P5

FL-3 CYC

488

667

P7

FL-4 APC

633

660

FACSCalibur Training
Spectral Overlap

Excited by a discreet wavelength from the excitation source

(laser), the fluorochrome will not emit a discreet wavelength
of light, but rather a spectrum, often a very broad spectrum.

FACSCalibur Training
Fluorescence and Spectral Overlap

Spectral Overlap

FACSCalibur Training
FITC Spectral Overlap into PE
FITC+ Control

FITC Overlapping into the PE Channel

Properly compensated

FL-2 (-) % FL-1 ~ 20-30%

FACSCalibur Training
PE Spectral Overlap into FITC

PE+ Control

PE overlapping into FITC, but not by much

Properly compensated

FL-1 (-) % FL-2 ~ 0.1-1.0%

Turning the Instrument On

FACSCalibur Training
Instrument Set up
Fluidics

Turn on the fluidics by pushing the green button that is located in the
middle right on the fluidics cart.

Check the fluidics status- make sure that there is an appropriate amount
of sheath fluid and that the waste container is not full. An alarm will
sound when there is a problem with either container.

Turn on the FACSCalibur by pushing the green button located on back

right of the instrument

If you are the first user of the day, turn the computer on or restart it if
left on from previous night.

When the computer restarts, click Administrator and then type in the
password (in all caps) BDIS. This will grant you access to the computer
and the BD Cellquest Pro software.

Allow a few minutes for instrument to warm up.

After instrument warms up, it will be ready for use.

FACSCalibur Training
Instrument Setup
Starting Up CellQuest Pro and Connecting to the Cytometer

Go to the bottom of the computer screen where the menu icons are
displayed. Click the BD CellQuest software icon which will allow the
software to load.

A new blank document will open. Click out of that and do not save
changes.

Go to Acquire> choose Connect to Cytometer. This will connect the

instrument to the computer.

Next, go to Cytometer> and choose Detectors and Amps,

Compensation, Threshold, and Status. These are the main palettes
that allow you to control the instrument. Using the status palette displays
if the instrument is ready for use.
Once the instrument is warmed up and ready for use, a QC procedure
called Time Delay Calibration must be performed. This will allow the
lasers to fire in their appropriate time in space. This must be done every
time a user turns on the machine.

FACSCalibur Training
Instrument Setup
Instrument Quality Control- Time Delay Calibration
1.
Go to File> Choose Open Document and then look in Data 1> Setup
Folder> and choose Time Delay Calibration.
2.
Next go to Cytometer> choose Instrument Settings and then look in
Data 1> Setup Folder and then choose IS Time Delay Calibration.
Next choose Set and then choose Done. This will load the
instrument setting for the Time Delay Calibration.
3.
Load the beads that will be provided with the label Time Delay Beads
and click Acquire.
4.
Make sure that the mean of the FSC peak is ~400 and the FL-4 peak is
~800 by adjusting the appropriate voltage on the Detectors and Amps.
5.
While the instrument is acquiring, go to Cytometer> choose Time
Delay Calibration and then when the message appears and choose
Ok.
6.
You will hear a sound coming from the computer acknowledging that
the Time Delay has been performed properly.
7.
The instrument is now ready for use.

FACSCalibur Training
Using the Instrument
Now the machine is ready for use. The QC has been performed and you
are now ready to use the instrument for your analysis needs. Located
below is a brief description of how to set up a protocol and how to label
your folders and file names.
Creating a New Protocol
1.
Go to File and choose New Document. A blank document will
appear.
2.
You will need to make plots to acquire your data. Go to Plot choose
Dot Plot. Go to Windows and choose Inspector. The inspector
will allow you to format your plot.
3.
With your plot highlighted and looking at the Inspector choose Plot
Type Acquisition to Analysis, choose your parameters (FSC vs. SSC,
or FL-1 vs. FL-2 etc) and make the appropriate number of plots that are
needed for your analysis. You can also make single parameter plots
called histograms and format them the same way that the dot plots were.
The plots you choose will solely be based on the analysis that you need.

FACSCalibur Training
Using the Instrument
4. You can also highlight a plot and go to Stats and then choose
Histogram or Quadrant stats based on the plot type. The software
allows you to edit the stat boxes to display any number of items that are
pertinent to you. Remember- the simplest experiment is the best
experiment.
5. Once you have everything in place and to your liking, go to File and
choose Save Document As and then navigate to your investigators folder
and give your protocol a name that you can remember and one that fits the
protocols needs e.g. FITC-PE, Cell Cycle, or Dendritic Cells with
DsRed or whatever best describes your experiment so you can remember
it.

FACSCalibur Training
Naming of Folders and Files

Naming of Folders and files is very important. This will

allow you to go to your Investigators folder and find your
data easily for future analysis.
Naming of Folders and Individual Files
1. Go to Acquire and choose Parameter Description. This
palette will allow you to control the instrument- naming of
folders, acquiring, and naming of individual files (Sample
ID)
2. On the Acquisition Palette look for the Directory Line and
choose Change. Navigate through Data 1> Sample Files
C1> and then look for your investigator and click on their
folder and then choose New Folder. Name the folder
C1ATAPR2407
FACSCalibur

PIs initials 3 letter month abbreviation

Today's date and year

FACSCalibur Training
Folder and File naming

Folder name=

C1WCAUG1108

File name=

C1WC081108

FACSCalibur Training
Naming of Folders and Files
3.

4.

Next, name each file. On the Acquisition Palette choose Change in

the File line. In the Custom Prefix line name the File C1AT042007
with the only difference from the Folder name being the number for the
month instead of the three letter abbreviation.
Go to File and then manually enter 1.
Everything is now in place for you to acquire your data. Take the time to
make sure that the folders and files are labeled correctly and that your
protocol is exactly the way that you want it. Making sure these things
are correct will save you time and frustration later.

FACSCalibur Training
Instrument Setup
In the careful planning of your experiment, you have determined that
controls are needed. Controls will allow you to set up the instrument
with regards to baseline fluorescence and compensation.
Negative and Single Positive Controls- Compensation
1.
In the setup mode, put on your negative or Ig control and click acquire.
2.
Place the negative in the first decade (100 101) on both axis of the dual
parameter plot or just on the x axis of the single parameter histogram.
3.
Once you have raised or lowered the voltage to get your negative in the
first decade, click off the setup box and click acquire. Based on your
storage and acquisition criteria, CellQuest Pro will save a file for you.
4.
Next, take a single positive control and make sure that you are in setup
mode. Put your tube on and click acquire. Adjust the compensation
accordingly to subtract out the % of the control tube out of the other
colors. e.g. If you are using a FITC single + control, you would use FL2-% of FL-1 or if you were using a PE single + control, you would use
FL-1 -% FL-2. In other words you are subtracting a percentage of one
color from another color.

FACSCalibur Training
Controls needed

Unstained

Single + Control for each fluorochrome used

Ex. If you have a 3 color experiment you need 4 total controls.

FACSCalibur Training
Instrument Setup
5. As a guideline- the FL-2 - % FL-1 is roughly 20-30%. The FL-1 % FL-2
is roughly 0.1-1.0%. However, these are just guidelines and vary based on
assay being performed, staining technique, antibody concentration, etc.
****You need to have single positive controls for all the colors you are using
including a negative. The more controls that you have the better your
experiment will turn out.
6. Once you have run all your controls you can proceed and acquire the rest of
your data.
7. After you are done with your run place a tube of water on the instrument
(sample injection port) and place the machine in standby. You are free to
go on about your day. Nothing else is required of you.

FACSCalibur Training
Shutting the Instrument Down
To shut the machine down:
1.
Take a tube of water and place on the instrument and run for 2 minutes.
2.
Take a tube of 10% bleach and run for 2 minutes
3.
Take a tube of water and run for 2 minutes and leave tube on instrument.
4.
Place instrument in standby mode.
5.
Turn the computer off by going into the menu and choose shutdown and
then proceed to turn the instrument off by pushing the green button
located in the back right, and then finally push the green button on the
fluidics cart.