Transcript Flow Cytometry Basic Training

FACSCalibur Training
A Look Inside the Box
Noor Khaskhely, M.D., Ph.D. (Operator)
Benjamin Chojnacki (Operator)
Flow Cytometry Core Lab
University of Toledo College of Medicine
Background of Flow Cytometry
SECTION I
The Many Parts of Flow
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Experimental design
Sample preparation
Flow Basics
Choosing the proper instrument
Setting up the instrument
Collecting the proper data
Data Analysis
Interpreting the data
Specific
Graphics presentation and publication
Applications
Sorting
Courses
Cytometry v. Flow Cytometry
Cytometry
Flow Cytometry
• Localization of antigen is
possible
• Poor enumeration of cell
subtypes
• Limiting number of
simultaneous
measurements
• No localization of
antigens
• Enumeration of subtypes
via cell/size/granularity/
markers
• Can look at numerous
parameters
simultaneously at high
rate
Uses of Flow Cytometry
It can be used for…
•
Immunophenotyping
DNA cell cycle/tumor ploidy
Membrane potential
Ion flux
Cell viability
Intracellular protein staining
pH changes
Cell tracking and proliferation
Sorting
Redox state
Chromatin structure
Total protein
Lipids
Surface charge
Membrane fusion/runover
Enzyme activity
Oxidative metabolism
Sulfhydryl groups/glutathione
DNA synthesis
DNA degradation
Gene expression
Medline Publications citing "Flow
Cytometry"
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The use of flow in research has boomed since the
mid-1980s
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Mechanics of a Flow Cytometer
• Cells in suspension are brought in single file past
• a focused laser where they scatter light and
emit fluorescence that is filtered and collected
• then converted to digitized values that are
stored in a file for analysis
Fluidics
Optics
Electronics
Mechanics of a Flow Cytometer
(Simplified)
Fluidics- Simplified Schematic
Sample
Tube
Waste
Tank
Sheath
Tank
Vacuum
Sheath
Pressure
(Constant)
Sample
Pressure
(Variable)
Line Pressure
Fluidics- Sample Differential
10 psi
10 psi
10 psi
10.2 psi
10.4 psi
10.8 psi
• Difference in pressure between sample and sheath
• This will control sample volume flow rate
• The greater the differential, the wider the sample core.
• If differential is too large, cells will no longer line up single file
• Results in wider CV’s and increase in multiple cells passing
through the laser at once » » » Faster cell analysis BUT loss of
resolution!!
300
280
G0/G1 CV= 2.42
Low pressure
Count
260
240
220
200
180
68.70
19.16
9.56
160
140
120
100
80
S phase
G0/G1
60
40
G2/M
20
0
High pressure
Count
0
340
320
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
0
1024
2048
FL3
3072
4096
GO/G1 CV= 7.79
74.85
0
1024
2048
9.12
15.84
3072
4096
FL3
Effect of Flow Rate on Sample Data Resolution
With increased sample pressure, flow rate increases resulting in a decrease in data
resolution.
Optics- Light Scatter
• When light from a laser
interrogates a cell, that cell
scatters light in all
directions.
• The scattered light can travel
from the interrogation point
down a path to a detector.
Optics- Forward Scatter
Laser Beam
Original from Purdue University Cytometry Laboratories
FSC
Detector
Interrogation- Forward Scatter
• Light that is scattered in the
forward direction (along the
same axis the laser is
traveling) is detected in the
Forward Scatter Channel.
• The intensity of this signal
has been attributed to cell
size, refractive index
(membrane permeability)
• Forward
Scatter=FSC=FALS=LALS
Laser Beam
FSC
Detector
Original from Purdue University Cytometry Laboratories
Collection
Lens
SSC
Detector
Optics- Side Scatter
Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter
Channel. The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg.
granules, cell inclusions, etc.)– Side Scatter=SSC=RALS=90° Scatter
Granulocytes
SSC
Lymphocytes
Monocytes
RBCs, Debris,
Dead Cells
FSC
Why Look at FSC v. SSC
Since FSC ≈ size and SSC ≈ internal structure, a correlated measurement between them can
allow for differentiation of cell types in a heterogeneous cell population
Optics- Fluorescence Channels
• As the laser interrogates the cell, fluorochromes on/in the cell
(extrinsic or intrinsic) may absorb some of the light and
become excited
• Fluorochromes leave their excited state and release energy in
the form of a photon with a specific wavelength, longer than
the excitation wavelength
• Emitted photons pass through the collection lens and are split
and steered down specific channels with the use of filters.
Optics- Detectors
• There are two main types of photo detectors used in flow
cytometry

Photodiodes
o

Used for strong signals, when saturation is a potential problem (eg.
FSC detector)
Photomultiplier tubes (PMT)
o
More sensitive than a Photodiode, a PMT is used for detecting small
amounts of fluorescence emitted from fluorochromes.
Optics- Fluorescence Detectors
Laser Beam
FSC
Detector
Collection
Lens
Fluorescence
Detector A, B, C,
etc…
Original from Purdue University Cytometry Laboratories, Modified by Benjamin Chojnacki
Optics- Filters
• Different wavelengths of light are scattered simultaneously
from a cell
 Need to split the light into its specific wavelengths in order to
measure and quantify them independently. This is done with
filters
• Optical filters are designed such that they absorb or reflect
some wavelengths of light, while transmitting others
• 3 types of filters
 Long Pass
 Short Pass
 Band Pass
 Dichroic
Optics- Long Pass Filters
Transmittance
• Transmit all wavelengths greater than specified wavelength
• Example: 500LP will transmit all wavelengths greater than
500nm
400nm
Original from Cytomation Training Manual
500nm
600nm
700nm
Optics- Short Pass Filter
• Transmits all wavelengths less than specified wavelength
Transmittance
 Example: 600SP will transmit all wavelengths less than 600nm.
400nm
Original from Cytomation Training Manual
500nm
600nm
700nm
Optics- Band Pass Filter
• Transmits a specific band of wavelengths
Transmittance
 Example: 550/20BP Filter will transmit wavelengths of light
between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20)
400nm
Original from Cytomation Training Manual
500nm
600nm
700nm
Optics- Dichroic Filters
• Long pass or short pass filters
• Placed at a 45º angle of incidence
• Part of the light reflected at 90º , and part of the light is
transmitted and continues on.
Detector 1
Dichroic Filter
Detector 2
Spectra of Common Fluorochromes
Common fluorochromes used for violet, blue, and red laser flow cytometry
From BD Multicolor Fluorochrome Reference Chart
Electronics- Photovoltaics
• Once the detectors collect photons of light, they convert them
to current
 A voltage pulse is created as cells pass through the laser
 As the cell passes into the laser, an event window opens
 More light is scattered as the cell moves into the center of the laser
(maxima)
 As the cell leaves the laser, less and less light is scattered
 After a set amount of time, the window closes until another object
enters the beam
• The current is processed into signal that is then converted into
a digitized value
Electronics- The Pulse
Time
Voltage Intensity
Pulse Area
Pulse Height
Pulse Width
Time
Digitization of the Pulse
Each pulse has a height (maximum voltage) and area. Area scaling can be performed to
assure that data is linear, allowing comparisons to be made between data sets
Electronics- Linear and Log
Amplifiers
• When the current exits the detector, it passes through either a
linear or log amplifier where it is converting it into the voltage
pulse.
• Intensity of the voltage can be adjusted by amplifying it on a
linear scale or converting it to a logarithmic scale
 The use of a log amp is beneficial when there is a broad range of
fluorescence as that may need to be compressed (this is generally
true of most biological distributions)
 Linear amplification is used when there is not such a broad range
of signals (e.g. in DNA analysis and calcium flux measurement)
BD FACSCalibur Usage
SECTION II
Calibur Platform
• The BD FACSCalibur is a modular flow cytometer designed for
cell analysis
 2 lasers
 488 (primary); detects FSC, SSC, and fluorescence
 633 (secondary); detects fluorescence
 Various protocols can be used for cell and DNA analysis
 Immuno-phenotyping (CD4, CD8)
 Apoptosis (PI and Annexin)
 Cell Cycle
 GFP analysis
 High-throughput drug screening
• Flexible system that is very easy to use and is very useful
Calibur Platform- Fluorescence
Detectors
• 488 Laser (Blue) has
3 parameters/filters
to choose from
FL-1; 530/30
FL-2; 585/42
FL-4; 661/16
• 635 Laser (Red) has 1
parameter/filter
FL-3; 670LP
FACSCalibur- Start-Up
• Fluidics should be started first
 Make sure there is an appropriate amount of sheath fluid and that
the waste container is not full
 Alarms will sound if there is a problem with either container during
startup
 Press the green button in the middle-right of the fluidics cart
beneath the FACSCalibur bench to start pump
• Flow cytometer should be started second
 Press the green button on the right side of the Calibur to turn on the
machine
• The computer should always be started last
 If the computer is already on, be sure to restart
 Login: Administrator
 Password: BDIS
• Always allow the instrument 15 minutes to warm-up prior to use
BD CellQuest Pro
• Once logon is complete, find the menu icons displayed at the
bottom-center and open BD CellQuest Pro
 A new blank document will open
• Go to Acquire> Connect to Cytometer
 Connects the instrument to the computer
• Go to Cytometer> Detectors and Amps, then return to the
Cytometer menu and choose Compensation, Threshold, and
Status
 These main palettes allow you to control the instrument
 The status palette will display if the instrument is ready for use
BD CellQuest- Quality Control
• Once the instrument has warmed up and ready for use, a QC procedure called “Time Delay
Calibration” must be performed
 Allows the flow cytometer to capture the signals at the right time from each laser as the cell
passes through them in succession
 Should be performed every time a user turns on the machine
• Go to File>Open Document browse to Data 1>Setup Folder
 Choose Time Delay Calibration
• Go to Cytometer>Instrument Settings, browse to Data 1>Setup folder
 Choose IS Time Delay Calibration
 Choose Set, then Done
 This will load instrument settings for the calibration
• Load calibration beads onto the bulk injection port and press Run on the front of the
cytometer
• Click Acquire, then go to Cytometer
 Choose Time Delay Calibration
 Select OK when message appears
 A sound will acknowledge that the operation was performed successfully
• Unload the calibration beads
• Press the Standby button on the front of the cytometer
• The cytometer is now ready for use (HUZZAH, etc.)!!!
BD CellQuest- Creating a New
Protocol
• If using a premade template, close the window that opened
when CellQuest launched
 If the window was closed inadvertently, go to File>New
Document
• In order to acquire data, plots need to be created
 Go to Plot and choose the appropriate type
 Go to Windows >Inspector
 This controls the formatting of the plots
Creating a New Protocol- Plot
Types
• There are several plot choices depending upon the
experiment’s application and data being captured
 Single Color Histogram
 Fluorescence intensity (FI) versus count
 Two Color Dot Plot
 FI of parameter 1 versus FI of Parameter 2
 Two Color Contour Plot
 FI of P1 versus FI of P2. Concentric rings form around populations.
The more dense the population, the closer the rings are to each
other
 Two Color Density Plot
 FI of P1 versus FI of P2. Areas of higher density will have a different
color than other areas
Creating a New Protocol- Plot
Types
Contour Plot
Density Plot
Greyscale Density
Dot Plot
Histogram
www.treestar.com
BD CellQuest- Creating a New
Protocol
• Once a plot type is selected, it will need to be formatted
properly
 With the plot highlighted choose Acquisition to Analysis from Plot
Type in the Inspector
 Choose the appropriate parameters (or parameter for histogram
plots) for the axes
• Repeat until there are an appropriate number of plots for the
analysis
 This will be based on the needs of each project
• Place gates in each plot to distinguish areas of interest
 Since there is no data, gates can be placed in the plot where data
is expected to be
 Gates can be adjusted during data acquisition
Creating a New ProtocolGating
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Gates are used to isolate subsets of cells or “populations” on a plot
Allows the ability to look at parameters specific to only that subset
Can use boolean logic to include or exclude multiple gates
For scatter plots there are 3 main types of gates:
 Rectangular
 Generally used to define a region of interest; can also be used to define a
broad population with outliers
 Polygonal
 Used define clustered/dense populations of interest
 Quadrant
 Most often used in multicolor to distinguish negative populations from
single- and double-positive ones
• For histograms bar/horizontal gates are used to define spikes/peaks
of interest
BD CellQuest- Creating a New
Protocol
• Once gates have been set around areas of interest, stats can
be displayed
 Highlight a plot and choosing Histogram or Quadrant (depends
on the plot type)
 Stat boxes can be edited to display any number of items pertinent to
the experiment/project
 Statistics can also be displayed for a plot without gates if needed
• Once the plots are in place, go to File>Save Document As
 Navigate to the appropriate investigator’s folder
 Give the protocol an appropriate name
Creating a New ProtocolNaming Folders
• When performing analysis a new folder should be made to
contain the data from that run
 Go to Acquire>Parameter Description
 Palette allows control of instrument, naming of folders and sample
(Sample ID)
 On the Acquisition Palette go to the Directory line and choose
Change
 In the navigation window go to Data 1>Sample Files C1>(PI’s Folder)
 Choose New Folder
 The new folder should be named to include:
 Date (including year)
 Your Initials
 A new folder should be made for each day of use
Creating a New ProtocolNaming Files
• The default name for each file can be changed if more
specification is needed
 On the Acquisition Palette go to File line and choose Change
 In the Custom Prefix line specify the name the files should have
 In the File line enter the desired suffix number of the first file
 This number will be incremented with each sample as data is saved
• Everything is prepared for acquiring data (HUZZAH, etc.)!!!
BD CellQuest- Controls
• Every experiment performed on the cytometer should include
controls
 Just plain, good ol’ fashioned science
 Distinguish cell populations from debris or background
 Allow differentiation of labeled cells (i.e., cell subtypes)
 Necessary controls will depend on the experiment
• Common controls include
 Single negative controls
 Unlabeled cells
 Isotype controls
 Fluorescence minus one (FMO)
 Single positive controls
 One for each color being used
 BD Comp Beads
BD CellQuest- Setting Up
Controls
• Start acquiring data with Negative or Isotype controls
 In the Acquisition palette check the Setup box next to the
acquisition controls
 Place the control tube on the bulk injection port
 Press the Run button on the front of the cytometer and click Acquire
 Adjust the voltage of the appropriate parameters
 If there is debris, adjust the threshold
 Data should placed so that it is easily viewable without going beyond
the 2nd decade
Setting Up Controls- Threshold
• When the laser interrogates an object, light is scattered.
• If the amount of light scattered surpasses a threshold, then
the electronics opens a set window of time for signal
detection
• The threshold can be set on any parameter, but is usually set
on FSC
• This is generally used to eliminate debris or cell fragments that
are not of interest
Setting Up Controls- Threshold
FSC
Detector
Threshold
(eg. 52)
Time
FSC
Detector
Threshold
(eg. 52)
Time
BD CellQuest- Setting Up
Controls
• After the FSC, SSC, and threshold are adequately set, abort
acquisition and uncheck the Setup box
 Begin acquiring data
 Data will be automatically saved based on the settings performed
• Once negative controls have been recorded, repeat the
process with single positive controls or FMOs
 While running in Setup mode, adjust the parameter voltages to
perform compensation
Setting Up Controls- Cell
Populations
• When adjusted
correctly, FSC and
SSC should
differentiate cell
populations
Granulocytes
Monocytes
Lymphocytes
Red Blood Cells/Debris
• Based on granularity
and size
Setting Up ControlsCompensation
• Fluorochromes typically fluoresce over a large part of the
spectrum (100nm or more)
• Depending on filter arrangement, a detector may see some
fluorescence from more than 1 fluorochrome. (referred to as
bleed over)
• Compensation needs to be made for this bleed over so that 1
detector reports signal from only 1 fluorochrome
Setting Up ControlsCompensation
FITC+ Control
• Parameter voltage
must be altered to
compensate for this
overlap
Proper compensation
used to be performed
visually, but this has
been found to be very
wrong
In order to properly
ensure compensation,
must use Mean
Matching
FITC Overlapping into
the PE Channel
FITC Properly
Compensated
Setting Up ControlsCompensation
• In the Compensation panel, locate the channels requiring
compensation (FL2-% of FL1, etc.)
 The positive control you are using is the first channel being
subtracted from the second (FITC from PE as above)
• Place the positive control tube onto the bulk injection port
check the Setup box and click Acquire
 Begin making small adjustments to the percentage
 After each adjustment, press Stop followed by Restart in either
the Parameter Description or Acquisition panel
 Watch the Mean Fluorescence Intensity (MFI) of the positive
control compared to the negative
 Continue compensating until the mean of the positive population is
nearly matched to the mean of the negative
Once the means have been approximately matched, abort
acquisition, uncheck the Setup box, and begin acquiring data
BD CellQuest- Performing Your
Experiment
• Once all controls are set and compensation has been
performed (if necessary), start running unknown samples
 Be sure that the Setup box is left unchecked
• Continue acquiring until all samples have been run
• If another appointment follows yours, do not shutdown the
cytometer
• Place a tube of water on the sample injection port and allow it to
run for ~2 minutes.
• Press the Standby button on the front of the cytometer
• You may now skip away in bliss at having successfully
revolutionized your field of research with your staggeringly
awesome data (DOUBLE HUZZAH)!!!
FACSCalibur- Shut Down
• If you are the last appointment of the day, once all samples are run,
place a tube of sterile water onto the sample injection port
 Keep the cytometer in Run mode
 Leave the tube on for ~2 minutes
• Replace the tube of water with the tube containing 10% bleach for
~2 minutes
• Place the tube containing water back onto the cytometer for
another 2 minutes
• Place the cytometer in Standby mode
• Turn the computer off
 Go to File>Shutdown
• Turn the instrument off by pressing the illuminated green button on
the back, right side of the cytometer
• Switch off the fluidics cart by pressing the illuminated green button
Wrap-Up
SECTION III
Interpretation
• Once the values for each parameter are in a list mode file,
specialized software can graphically represent it.
• The data can be displayed in 1, 2, or 3 dimensional format
• Common programs include…



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
CellQuest
BD FACSDiva
Flowjo
FCS Express
BD Paint-a-Gate
• Every program has its Pros and Cons, most require licensing
 Paint-a-Gate is available on the workstation in the Flow Core
Important Points on Analysis
• What kind of data are you looking for?




How much fluorescence?
What percent are positive?
How much more positive is x than y?
What is the ratio between param1 and param2
• What kind of statistics are available





MFI (geometric or arithmetic)
%-ages
CV
Median
Anything you can do with a list of numbers
Everything’s Relative
• The relative bin numbers are just that…relative.
• Saying your cells have a mean fluorescence intensity of 100
means absolutely nothing until you compare it to a negative.
• The fact that everything is relative allows you to compare 2, 3,
or 20 samples using the same instrument settings.
FACSCalibur- Troubleshooting
• Under normal weekday circumstances, the cytometer should be
ready for use
 Contact one of the operators to change the sheath or empty the
waste
• If using the instrument on the weekend, set up will need to be
performed
 If the sheath is empty, Refill the sheath from one of the BD FACSFlow
containers designated
 If the waste is full , empty the container into the sink and reconnect
the cap/tubing
• If any issues with the cytometer are encountered:
 DON’T PANIC!! (Always carry a towel)
 Remove any sample running
 Place the cytometer in Standby
 Contact one of the operators
• Do not try to fix the instrument under any circumstance
 We have a service contract for that 
Appointments
• Flow appointments should be scheduled through one of the
Flow Core operators
• No appointments by email
 Call Ben @ x4277
 Stop by HEB 270 or 211
• Please arrive on time
 Billing begins at the time scheduled
 Additional time for use will be allowed only if no other users are
scheduled following the appointment
• Billing is done in 15 minute increments
• Cancellations should be made at least 24hrs in advance
• Cancellations without proper notice will be billed accordingly
unless there are exigent circumstances
References
• Numerous References available in the Flow Lab
 Cytometry
 Current Protocols in Flow Cytometry
 Many more reference books available
• Purdue University Cytometry Laboratories website:
http://www.cyto.purdue.edu/
 Dr. Robert Murphy, Carnegie Mellon University- Basic Theory 1
and 2 PowerPoint slides
• The Scripps Research Institute Flow Cytometry Core Facility:
http://facs.scripps.edu/
Flow Lab Contact Info…
• Noor Khaskhely, M.D., Ph.D., Flow Operator
 [email protected]
• Benjamin Chojnacki, Flow Operator
 [email protected]
 x4277