BD FACS Canto II Flow Cytometer
User Operations Guide
Table of Contents:
1. Instrument Specifications ............................................................................................ 2
2. Safety Considerations ................................................................................................. 3
3. Instrument Access ....................................................................................................... 3
4. Supplies provided for the instrument ........................................................................... 4
5. Supplies to obtain for your experiment ........................................................................ 4
6. Things to consider in advance ..................................................................................... 5
6.1 General .................................................................................................................. 5
6.2 Fluorophore selection ............................................................................................ 5
7. How to operate the BD FACSCanto II ......................................................................... 5
7.1 Startup procedures ................................................................................................ 6
7.2 Running a CS&T Performance Check ................................................................... 6
7.3 Starting an experiment – setting instrument parameters ....................................... 7
7.3.1 Compensation ................................................................................................. 7
7.4 Running samples ................................................................................................... 8
7.4.1. Global vs. Normal Worksheets ....................................................................... 8
7.5 Shutdown procedures ............................................................................................ 9
8. Maintenance Methods ................................................................................................. 9
8.1 Changing fluids ...................................................................................................... 9
8.2 Emptying the waste jug ........................................................................................ 10
9.Troubleshooting ......................................................................................................... 10
Please note that this guide is meant to reinforce your training session, and only contains the
minimum steps required for collecting a basic dataset. Consult the instrument manuals for more
information on the more advanced features of this unit.
All manuals must be left by the instrument after use.
Cytometer serial #: V96101177
BD Biosciences tech support: 877-232-8995
Field Service Engineer: Jared Jiminez (jjiminez@cytekdev.com)
Issues with the instrument: Angelo Benedetto, 713-348-8207 (office); 281-686-4924 (cell)
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last revision: 23 July 2013 AFB
1. Instrument Specifications
Manufacturer: BD Biosciences
Model: FACS Canto II (RUO special edition)
Flow cytometer with 2-laser, 6-color (4-2) configuration. Uses blue (488 nm) and red (633 nm)
lasers to excite fluorescent objects in the flow cell. FACSDiva software provides quantification
of cell populations, based on fluorescence emission and forward (FSC) and side (SSC)
scattering, related to cell size and shape (granularity).
Lasers:
- Blue (488 nm) laser and red (633 nm) laser. Violet laser option (407 nm) was not
purchased with this system.
Detection filters: (see attached diagrams of collection optics)
Excitation
Laser (nm)
488 (blue)
633 (red)
PMT
position
A
B
Dichroic
mirror
735 nm
655 nm
Filter
designation
780/60
670 LP
Collected
signal
750-810 nm
> 670 nm
Intended
dyes
PE-Cy7
PerCP,
PerCP-Cy5.5
Other dyes
C
D
E
(N/A)
556 nm
502 nm
585/42
530/30
564-606 nm
515-545 nm
PE
FITC
PI
GFP, CalceinAM
F
A
B
C
(none)
735 nm
(N/A)
(none)
488/10
780/60
483-493 nm
750-810 nm
SSC
APC-Cy7
660/20
650-670 nm
APC
PI, PE-Cy5.5,
7-AAD
APC-H7
AlexaFluor
633
Collection Modes:
- log, linear scales
Supported file formats:
- FCS
- FCS3 (compatible with FlowJo)
FlowJo analysis software is also available for advanced analysis of data from the
FACSCanto II.
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2. Safety Considerations
The BD FACSCanto II uses two enclosed lasers for sample analysis. These can be hazardous
if users look into the laser beam. Do not attempt to dismantle or adjust the laser hardware in
any way.
Primary human cells, taken directly from patients, are considered a BSL level II biohazard. The
flow cytometer creates an aerosol from cell suspensions, and these can be potentially
hazardous. Users must take appropriate precautions to prevent contamination of themselves
and other users with potentially harmful pathogens. Primary human cells which have not been
tested for communicable diseases must be permanently fixed (e.g. with an aldehyde-based
crosslinking agent) prior to use on the instrument.
IMPORTANT: ALWAYS REMOVE GLOVES BEFORE TOUCHING
THE KEYBOARD, MOUSE, OR COMPUTER!
3. Instrument Access
This instrument is available through the Rice Shared Equipment Authority (SEA@BRC).
Researchers can become users of the instrument by following these guidelines:
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All users must complete a training session from the instrument manager. New users
may not receive training from an existing user.
Users must log in using the login and password provided to them during training.
Anyone found using the instrument under another user’s login will have their usage
privileges revoked. Users must log off at the end of their session.
Instrument time must be reserved using the SEA Calendar. Any user reserving time on
this calendar will take priority over a walk-on user.
Users must be registered through SEA, with a valid fund and org number (Rice users).
Users must complete all required SEA paperwork before access will be provided to the
instrument.
Safety guidelines must be followed at all times. Users must remove gloves before
touching the keyboard, mouse, or computer. Users must use caution with hazardous
materials (e.g. human-derived cells), and follow documented biosafety methods.
Internet access will not be available for the instrument, since the computer and
cytometer use ethernet for communication, and because we wish to avoid malware
infections on the computer. Please bring a USB drive to retrieve your data. Software
may not be installed on the instrument computer without manager approval.
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4. Supplies provided for the instrument
- calibration beads for CS&T (stored in the Qutub refrigerator)
- all necessary sheath and wash fluids for the fluidics cart
- extra sheath fluid for preparing CS&T beads
- 10% bleach solution for cleaning the instrument
- full bleach for the waste jug
- paper and toner in the laser printer for any printouts
- please inform Angelo if any supplies are running low
5. Supplies to obtain for your experiment
- your cells, in standard 12x75mm flow cytometry tubes, filtered and pre-stained as
necessary
- pipettors and tips for resuspending your sample prior to analysis*
- waste container for your tubes and pipettor tips
- your USB drive for data retrieval
- gloves if needed
* please do not rely on the host lab to provide gloves, tips, reagents, or other consumables for
your experiment!
You may also want to consider the following items for your experiments:
- BD CompBeads: these beads will capture your labeled antibody of interest, and thus
serve as an automatic positive control for your experiments. This is particularly useful
for establishing compensation controls, and may also be useful in visualizing a “positive”
sample on the cytometer. 6 mL of CompBeads costs ~$225, and requires only one drop
per test.
- BD Calibrite Beads set: these fluorescent beads are useful for learning flow cytometry,
and practicing methods like compensation and sample analysis.
- 35 µm filter tubes (e.g. BD# 352235) – these provide a filter in the snap-on cap for each
tube, making filtering easy.
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6. Things to consider in advance
6.1 General
- Cell analysis by flow cytometry usually falls under three categories: (1) unstained
analysis of cells for size and granularity (e.g. clinical analysis of various cells in blood);
(2) intra- and extracellular staining of cell protein markers using fluorescently-conjugated
antibodies; (3) fluorescent staining of cells without antibodies (e.g. live/dead analysis via
calcein-AM/EthD-1; cell cycle analysis via PI).
- A flow cytometer will enable fast analysis of a large number of samples, and the
dedicated software gives users a view of dozens of combinations for cell expression of
each marker. However, the majority of user issues with data analysis and interpretation
will be due to incorrect sample preparation (i.e. staining methodology) or planning for
controls. Data from flow cytometry will only be as good as your sample, so spend the
necessary time to plan out all aspects of your experiment before beginning!
- Flow cytometry relies on a large number of “events” (usually 10,000) to provide a
believable analysis of cell phenotype. However, many more cells than this are usually
needed to be able to establish all positive and negative controls for compensation. Plan
your full matrix of antibody stains in advance to be sure that you have a sufficient
number of cells to complete a full analysis.
- Cytometry relies on a single cell suspension for cell analysis. Be sure to vortex your
sample prior to placing it onto the analysis arm. “Sticky” cells may otherwise appear as
doublets or as larger clumps.
6.2 Fluorophore selection
- Note that the fluorescent dyes used in flow cytometry can be sensitive to room lights,
and may lose their fluorescence substantially upon exposure. Tandem dyes (e.g. PECy7, or PerCP-Cy5.5) are particularly vulnerable to this. (CS&T beads are also
susceptible – preparations of CS&T beads in sheath fluid, left open to room lights, will
fade sufficiently over the course of an hour that they will fail the CS&T instrument check.)
Keep your samples covered, and on ice!
- Check for overlap between or among your different fluorophores. For example, FITC
emission will leak over substantially into the PE channel, yielding incorrect data. This
can be addressed in two ways: (1) If possible, choose fluorophores with well-separated
emission spectra. (2) Always run compensation controls when using multiple
fluorophores, regardless of their separation. These controls require running your
brightest, single-labeled versions of cells for each fluorophore (i.e. FITC alone, PE alone,
Cy5 alone, etc.). BD CompBeads can be used for this purpose – they can be stained for
nearly any fluorescently-tagged antibody, providing your positive compensation controls
without using your precious cells. FACSDiva software also makes the compensation
process easy, by establishing the necessary individual positive controls, and then
calculating and applying the compensation to your samples. For any multi-fluor
experiment, compensation should be a part of your plan.
7. How to operate the BD FACSCanto II
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IMPORTANT: ALWAYS REMOVE GLOVES BEFORE TOUCHING
THE KEYBOARD, MOUSE, OR COMPUTER!
7.1 Startup procedures
- The BD FACSCanto II is located in BRC 610H. Users can receive laboratory card
access upon completion of their training session. Contact Gloria Funderburg
(gloriaf@rice.edu) for details.
- Before turning on the instrument, check the three fluid boxes and the waste jug on the
fluidics cart under the cytometer. If any one of the fluid boxes feels less than 25% full,
change it out with a full container as described below (8.1). If the waste jug feels more
than half full, empty it out as described below (8.2).
- The cytometer will usually be shut down from the previous user. If so, turn on the
cytometer main power. The green power button is on the left side of the cytometer and
powers on both the cytometer and the fluidics cart. It’s normal to hear sounds from the
fluidics cart as it starts up.
- Turn on the PC and log in using the login and password. Start FACSDiva and enter your
individual username and password. If this screen does not appear immediately, resize
the window slightly to force it to redraw.
- Ensure that the software is connected to the cytometer by checking the bottom of the
Cytometer window. (this may take a few moments. If it doesn’t connect, select
Cytometer > Connect and inform the lab manager.)
- Recheck fluid levels in the Cytometer window (bottom right) and contact the lab manager
if levels are low.
- Run Fluidics Startup by selecting Cytometer > Fluidics Startup. This can take about 10
min, and the workspace will indicate “Fluidics Startup done” in the lower right corner
when complete. (The “warm-up time” indication in the Cytometer window refers to the
lasers. The fluidics startup can be completed while the lasers are still warming up.)
7.2 Running a CS&T Performance Check
This procedure should be performed once each day, before running any experiments. The
CS&T check generates default settings within the optimal detection range for the PMTs, adjusts
the laser delay times as needed, and ensures that the cytometer is performing stably over time.
Completing a daily CS&T also allows your data and sampling conditions to be transferred to any
other BD cytometer running FACSDiva (e.g. a sorting system like FACSAria). Check the log
book or the CS&T window to determine if this check has already been completed for the day.
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Prepare CS&T beads by adding one drop of CS&T beads (stored in 4°C refrigerator) to
350 µL of sheath fluid. Be sure to shake the vial (resuspending the beads) first!
Select Cytometer > CST to begin. A separate CS&T software package will open, and
the cytometer will disconnect from FACSDiva and connect to the CS&T software.
Check the lot ID under Setup Beads, and ensure that this matches the lot ID on the
beads vial. (If not, contact the lab manager. The profile for each lot ID must be
downloaded from BD and installed on the PC.)
Install the CS&T bead tube on the SIT and click Run. CS&T takes about 5 min.
Remove the tube from the SIT when the performance check is complete. Verify that the
cytometer passed the CS&T check. Contact the lab manager and record in the log book
if the cytometer does not pass.
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Select File > Exit. CS&T will disconnect from the cytometer, close, and return control to
FACSDiva.
If a CS&T mismatch dialog appears, select “Use CST Settings”.
7.3 Starting an experiment – setting instrument parameters
All of your measurements within FACSDiva are conducted in the context of Experiments within
the Browser window. To help in keeping your Experiments organized, you can create Folders to
store these Experiments. Each Experiment must be opened before it can be used, and each
contains its own set of Settings, and multiple Specimens and Tubes.
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If you prefer to keep your experiments organized in folders, you can begin by clicking the
“New Folder” icon in the Browser folder
Click the “New Experiment” icon to create a new Experiment. The Experiment is
automatically opened (the open book icon), and any other Experiment is closed. Only
one Experiment may be open at a time.
Click the Cytometer Settings icon in the Browser. The Inspector window will show all
possible fluorophore channels (FITC, PE, APC, etc.) in the Parameters tab. Select
individual channels and click the Delete button if they will not be used in your
experiment. Repeat for each Parameter until you only see FSC, SSC, and the other
fluorophore channels used in your experiment.
7.3.1 Compensation
- If using more than one fluorophore, select Experiment > Compensation Setup > Create
Compensation Controls... The window that appears should contain each of the
fluorophore channels that you previously selected in the Parameters tab. If desired, you
can type the name of each antibody in the Label field (replacing the “Generic” text), e.g.
“CD44” if you’re using FITC-CD44 antibody.
- Click OK. FACSDiva will add a Specimen to your Experiment called Compensation
Controls. Inside of this Specimen are Tubes (i.e. individual samples) for your Unstained
Control, and each single-stained Control. Be sure to have each of these control samples
prepared before you start.
- Click on the arrow to the left of the Unstained Control Tube. The arrow should turn
green. Load the Unstained sample and click the Acquire Data button in the Acquisition
Dashboard. Wait a few seconds until events begin appearing in the Normal Worksheet.
Choose a suitable Flow Rate (medium is usually a good starting point).
- The majority of events should appear in the center of the FSC vs SSC graph in the
Normal Worksheet. If this is not the case, click on the Parameters tab in the Cytometer
window. Click on the Voltage slider for FSC and adjust this up or down until new events
appear in the center of the graph (usually within the preset P1 gate). Click Restart in the
Acquisition Dashboard if new events are difficult to see. Repeat for the SSC Voltage.
When the majority of new events are completely centered within the P1 gate, click the
Record Data button in the Acquisition Dashboard.
- After all Events have been recorded, the cytometer will stop, and the SIT arm will drop
down. Remove the tube, and allow the SIT to rinse.
- Click the Next Tube button in the Acquisition Dashboard and place the first singlestained fluorophore (e.g. FITC) onto the SIT. Click the Acquire Data button and begin
collecting events. Your samples should fall within the same P1 gate on the FSC-SSC
graph. The adjacent graph will show where your positive control samples sit on their
fluor graph (e.g. FITC vs Counts). These positive samples should be much higher than
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your unstained controls. Check back to the Unstained Control tab to compare (e.g.
Unstained FITC controls might be centered at 102, while positive controls might be
centered at 104-105. If this is the case, click Record Data and collect the necessary
number of Events.
Repeat the above step for each single-stained control. If necessary, you can adjust the
Voltage parameters for the fluors in the Parameters window, to put individual fluors on
axis. However, note that this will disrupt the Compensation process, and will require
starting over from the Unstained Control and re-recording each Control sample.
After all Control tubes have been recorded, select the Compensation Control specimen
and in the menus choose Experiment > Compensation Setup > Calculate
Compensation... Give the Compensation a name and choose Link&Save. The
Cytometer Settings icon under your Experiment will have a small chain-link icon. In the
Cytometer window, the Compensation tab will indicate the Spectral Overlap percentage
of each fluorochrome.
7.4 Running samples
- Create a new Specimen in your experiment, and create any necessary plots on the
global worksheet (see 7.4.1. for Global vs Normal worksheets). You’ll want to create at
least a FSC vs SSC plot.
- Create a new Tube under the current Specimen, and click on the arrow to its left so that
the arrow becomes green. Vortex your sample briefly, and install it onto the SIT.
- Click Acquire in the Acquisition Dashboard window, using medium speed. As the
instrument begins acquiring, assess the number of events per second; 100 cps is usually
a reasonable rate for acquiring data. Adjust the speed if a higher or slower rate is
needed. (If the events/second is too low even at a high rate, then you may need to
prepare your samples at a higher cell concentration in the future.)
- Use the FSC and SSC voltage controls in the Cytometer window to center your
population of cells on the FSC vs SSC graph. Note that most cell suspensions have
some amount of cell debris (which will appear as very small objects), and some amount
of doublets or multiple clumped cells (which will appear as very large objects). Do not
mistake these for your real cells!
- When adjusting the voltages on each channel, click Restart to clear the current data and
begin observing a fresh set of data using the new parameters.
- When your cells are well-centered and are being Acquired, click Record to record the
data. The parameters at the bottom of the Acquisition Window will determine how many
events (total, or within a given gate, or under other criteria) will be recorded before the
instrument stops and ejects the sample.
7.4.1. Global vs. Normal Worksheets
- Note that the Worksheets on the right side of the screen come in two flavors: Global and
Normal. The icon in the upper left corner of the Worksheet window will switch between
the two.
- Global worksheets contain elements (graphs, etc.) that appear for every tube. In most
cases, these will be what you’ll use.
- Normal worksheets contain elements that are specific to one individual tube. These
allow for custom additions of specific graphs or tables to a worksheet.
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7.5 Shutdown procedures
- Save your data! Copy your data to a USB drive before leaving. We cannot guarantee
the safety of any data on the computer.
- Run a cleaning tube
o Open a new experiment in the Browser, and set the current tube pointer to a
tube.
o Install a tube containing 10% bleach solution on the SIT.
o Acquire for 5 min, then remove the tube from the SIT.
- If another user will be coming within 2 hours, do not shut down the system, as laser
lifetime is degraded by frequent startup/shutdowns. Instead:
o Select Cytometer > Standby, and choose the Disconnect from Cytometer Only
option to reduce the fluid flow.
o Select File > Log Out and select Log Out Only.
- If no other user will be coming within 2 hours:
o Run Fluidics Shutdown by selecting Cytometer > Fluidics Shutdown. Do not exit
the FACSDiva software or shut down the cytometer before this step is complete,
or you may damage the cytometer!
o After fluidics shutdown is complete, and FACSDiva indicates that the system can
be turned off, turn off the cytometer using the power button on the left side.
o Wet a kimwipe with DI water and gently wipe the SIT, then dry with a fresh
kimwipe.
o Exit FACSDiva software and shut down the PC.
8. Maintenance Methods
8.1 Changing fluids
If any of the FACSFlow, FACSClean, or FACSShutdown boxes feel < 25% full, change them out
using the following procedure.
- Ensure that the cytometer is not acquiring events. Pull the fluidics cart out from under
the cytometer to gain better access to the fluidics boxes.
- For each empty fluid, detach its sensor and fluid line from the cart by gently pulling the
sensor straight out, and pressing the metal clip on the fluid line to release the tubing.
Never leave the sensor line attached to the cart when changing fluids. The sensor
wires are fragile and can break if bent.
- Unscrew the cap on the fluid box, lift out the sensor assembly, and set aside.
- Place a new fluid box on the fluidics cart. Open the box at the perforated opening.
Remove the cap, and pull the plastic ring around the opening to lift out the ring. Place
the cap on the old fluid box and save the unused fluid for later use.
- Insert the sensor assembly into the new fluid box, and hand-tighten the screw until it is
fully closed.
- Reattach the sensor line to the fluidics cart by aligning the connection and inserting
directly. To attach the fluid line, push the coupling into the port until it clicks.
- Prime the fluidics by selecting Cytometer > Cleaning Modes > Prime After Tank Refill.
Only select the fluid that you changed.
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8.2 Emptying the waste jug
If the waste jug feels > 50% full, empty it out using the following procedure. Use proper
precautions, as the waste container will contain 10% or greater bleach solution, along with any
biological samples analyzed on the cytometer. Use appropriate protective clothing, eyewear,
and gloves.
- Ensure that the cytometer is not acquiring events.
- Detach the sensor and fluid line from the cart by gently pulling the sensor straight out,
and pressing the metal clip on the fluid line to release the tubing. Never leave the
sensor line attached to the cart when changing fluids. The sensor wires are
fragile and can break if bent.
- Carefully move the waste jug to the sink. Do not slosh the liquid inside or get liquid onto
the red waste vent, as this will prevent the cap from functioning properly.
- Unscrew the large waste cap and trap (the beige plastic cap) from the jug and set aside.
- Empty the bleach-exposed waste into the sink. Rinse the sink for several minutes with
tap water to flush the waste contents.
- Add approximately 1L of bleach to the empty 10L waste jug. This is approximately 1
inch of bleach solution in the bottom of the jug.
- Reattach the large waste cap to the jug, and return the jug to the fluidics cart.
- Reattach the sensor line to the fluidics cart by aligning the connection and inserting
directly. To attach the fluid line, push the coupling into the port until it clicks.
9.Troubleshooting
9.1 Check the BD Troubleshooting sheet?
9.2 I can’t see the cytometer – it says that it’s disconnected
- recheck the network cables, make sure they’re plugged in fully
- Select Cytometer: Connect to attempt to connect
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