UNIVERSITY OF CALGARY
FLOW CYTOMETRY FACILITY
‘Efficient and reliable flow cytometry
services with the highest standards of
quality and productivity’
Flow cytometry is a technique for measuring physical and
chemical properties of individual cells as they travel in
suspension one by one past a sensing point.
BASICS OF FLOW CYTOMETRY
•
The cells in suspension are forced to pass in a
fluid stream through a flow cell.
•
The fluid stream intersects the focus of a laser.
•
The laser light is scattered and, if the cells are
fluorescent, they produce fluorescent signals.
•
These light signals are then
electronic signals (voltages).
converted
to
INSTRUMENTATION
1. Fluidics
2. Optics
3. Electronics
FLUIDICS
•The purpose of the fluidics system is to transport
cells in a fluid stream to the laser beam for
interogation.
OPTICS
The optics system consist of a laser to
illuminate the cells in the sample stream and
optical filters to direct the resulting light
signals to the appropriate detectors (I.e . to
resolve different colors).
1. The flow of sheath fluid restricts the cells to the
center of the sample core for optimal illumination
(hydrodynamic focusing).
2. Only one cell should move through the laser beam at
a given moment.
“THE LASER”
• Lasers emit coherent light, in a fine, straight beam at a
specified wavelength.
• The use of a laser allows the beam of light to be
focused on single cells so that basic measurements
based on beam disturbance can be taken (FSC, SSC) .
LASER: Acronym for
Light Amplification by
Stimulated Emission of
Radiation
ELECTRONICS
• Processing of signals from detectors
-Cells passing through the laser beam generate light
signals.These light signals are then converted to
electronic signals (voltages).
-The electrical voltage generated will be proportional to
the number of photons (amount of light) emitted by the
cell/particle.
-The voltages are processed by the computer.
The sample is injected into a stream of sheath
fluid within the flow chamber; the sample core
remains separate but coaxial within the sheath
fluid.
•Light is bent (diffracted) depending on the size and
refractive index of the cell
•Detected along the axis of incident light
(0-100)
•Light is reflected/bounced to the side
•Proportional to cell granularity
•Detected at 90o to incident light axis
When cells pass through the laser intercept, they
scatter laser light and the system measures the
degree and direction of scattered light-indicators
of the cell’s size, shape and structure.
What do the scatter signals tell us?
•
Together the forward and side scatter signals can
provide useful ways to characterize different cell
types.
• Example: Leucocytes (white blood cells).
FLUOROSCENCE
•Fluorescent compounds absorb light energy over a
range of wavelengths, which causes an electron in the
compound to be raised to a higher energy level.
•The excited electron emits this excess energy as a
photon of light.
•While each fluorochrome will have an optimal, or peak
emission wavelength, the spectra will actually be
distributed over a number of wavelengths.
Any fluorescent molecules present
on the cell fluoresce.
Injector
Tip
Sheath
fluid
Fluorescence
signals
Focused laser
beam
•
The cells are forced to pass in a fluid
stream through a flow cell where the fluid
stream intersects the focus of the laser.
Detector
Fluorochrome
Emission
FL1
FITC, GFP, Alexa
488, CFSE, Fluo 3
525 nm
FL2
PE, PI
575 nm
FL3
PER-CP, Cychrome,
7-AAD
660-675 nm
More than one fluorochrome can be used simultaneously if
each is excited at 488 nm and if the peak emission
wavelengths are not extremely close to each other.
DATA DISPLAY
Negative Control
Sample
Histogram: Relative fluorescence vs. # of events
DATA DISPLAY
Quadrants can be applied to any 2 parameter
display.
IMMUNOPHENOTYPING
Antibody-fluorescent dye conjugates bind to
antigens and the quantity of the fluorescent light
emitted is correlated with the cellular marker in
question.
DATA DISPLAY
Activation: Surface Receptor Expression
Up Regulation of IL-2 Receptor on Mouse B Cells
CELL ADHESION MOLECULES
A. Unstimulated
--- Isotype
__ Anti-P-Selectin
B. Thrombin-activated
Expression of P-selectin is up-regulated on activated
peripheral blood platelets.
Activation: Intracellular Cytokines
INTRACELLULAR CYTOKINE MEASUREMENT
INTRACELLULAR CYTOKINE MEASUREMENT
Multi-parameter flow cytometric analysis of cultured Th1 and Th2 cells
APOPTOSIS
METHODS FOR DETECTING APOPTOSIS:
Annexin V assay
Apoptosis: Annexin V Assay
Jurkat Cells Treated for 6 hours with IgM Anti Fas Antibody
Annexin V
PI vs. ANNEXIN
• One of the more common uses of DNA-based dyes
is to identify apoptotic cells.
• Necrotic cells are widely permeable to a number
of cell labels (usually PI) whereas apoptotic cells
are impermeable.
• Staining for apoptotic markers (i.e., Annexin V)
will identify apoptotic cells, whereas necrotic cells
will also stain with PI.
PI vs. ANNEXIN
Apoptosis: Quantitative Analysis of Caspase-3
Activation
Jurkat cells Treated with Campothecin
Caspase 3 PE
Apoptosis: TUNEL Assay
Jurkat Cells Treated for 6 hours with IgM Anti Fas Antibody
APO-BRDU
0 Hours
6 Hours
Apoptosis: bcl-2 Regulation
Mitochondrial Protein bcl-2
Blocks Apoptosis
Bcl-2 Is Down Regulated
During Apoptosis
METHODS FOR DETECTING APOPTOSIS:
Gene regulation
Analysis of p53 expression in SV40 transformed rat ovarian cells
METHODS FOR DETECTING APOPTOSIS:
Histone Phosphorylation
Proliferation: Nucleotide Analogs
Bromodeoxyuridine Is
Incorporated Into
Cellular DNA
By Pulsing Proliferating
Cells
The Nucleotide May Be
Conjugated With
Fluorochrome
Or Detected By
Antibodies
PROLIFERATION: CFSE
Carboxyfluorescein succinimidyl ester
The amount of CFSE in the cell in the
membrane of proliferating cells
halves with each successive
division and therefore, the fluorescence
can be used to monitor the number of
cell divisions.
Ploidy: Nucleic Acid Dyes
DAPI is one of many
non-vital dyes that binds
DNA on an equimolar basis
This allows the precise
quantitation of a cell
populations proliferative
index
DNA & CELL CYCLE ANALYSIS
PI: Key feature of DNA probes is that they are
STOICHIOMETRIC.
Total nuclear content and the fraction of cells in each
phase of the cell cycle can be measured.
Normal Cell Cycle
G2
M
G0
DNA Analysis
G1
G0G1
s
C
o
u
n
t
G2 M
s
0
200
400
600
4N
2N
DNA content
800
1000
INTRACELLULAR CYTOKINE MEASUREMENT
• The production of cytokines by specific cell types
can be determined as opposed to measuring the
amount of secreted cytokine present in the serum
or supernatant.
APOPTOSIS: SUBGENOMIC DNA PEAKS
TRANSFECTION EFFICIENCY
Using Green Fluorescent Protein (GFP) as a cotransformation marker is one of the most
common applications of GFP-expressing vectors
CELL NUMBER
TRANSGENIC
CELL LINE
CONTROL
0
100
200
300
400
500
FLUORESCENCE
600
700
ESTIMATING CELL VIABILITY
PROPIDIUM IODIDE (PI)
-Excluded by viable cells and when taken up by dying
cells, binds to nucleic acids and fluoresces orange.
Viability: Fluorescein Diacetate & PI
FDA converted to fluorescent compound in live cells
Oxidative Burst
Conversion of nonfluorescent dichlorofluorescein diacetate to
the fluorescent compound 2',7'-dichlorofluorescein can be used
to monitor the oxidative burst in polymorphonuclear leukocytes.
CALCIUM FLUX
MEASUREMENTS
Many stimuli can cause mobilization of calcium either as
an influx from the extracellular medium or the release
of intracellular stores.
A number of dyes that fluoresce when bound to calcium
are used to “pre-load” cells prior to calcium mobilization
and the cells will “glow” as calcium streams into the
cells.
Fluo-3 is a Ca+2 sensitive fluorescent probe. Its emission
intensity changes with the Ca+2 concentration.
CALCIUM INFLUX
Ca++ Bound Indo-1 at 390 nm to Free Indo-1 at 495 nm
PHOSPHOPROTEIN PROFILING WITH
FLOW CYTOMETRY
Signal Transduction: Phospospecific mAbs
Signal Transduction:
Phosphospecific mAbs
PHAGOCYTOSIS
BD QUANTIBRITE SYSTEM
A method used to calculate the amount of
antibody bound to a cell that correlates to the
number of antigens expressed on the cell
surface.
BD CYTOMETRIC BEAD ARRAY SYSTEM
WASH
1. Add unknowns or standards to
capture bead array
2. Add detection reagents and
incubate
3.Acquire samples
Cell Sorting
• The population of interest can be separated during
flow and deposited into a tube for later analysis
(>95% purity).
• Any combination of analytical parameters can be
used to set the criteria for sorting as opposed to
single parameter methods (I.e. separating the
negatives from the positives).
Fluorescence Activated
Cell Sorting
488 nm laser
FALS Sensor
Fluorescence detector
Charged Plates
Single cells sorted
into test tubes
-
+
TRANSGENIC
CELL LINE
CELL NUMBER
P o s t S o rt 2
R2
POST-SORT
M1
R3
0
100
200
300
400
500
FLUORESCENCE
H is to g ra m S ta tis tic s
600
700
ADVANTAGES
-Evaluating large number of cells quantitatively and
reproducibly increases the statistical confidence and
precision of data.
-The ability to measure several parameters on thousands
of single cells within minute (10,000 cells/second).
-Measurements are made separately on each cell.
-Simultaneous, multiparameter analyses in complex cell
populations.
The power of flow cytometry lies in the ability to
measure several parameters on thousands of
single cells within minutes
(1000 cells/second).
BD FACScan is a 3-colour, fixed alignment
bench top analyzer, equipped with a 488 nm
air-cooled argon laser. It is capable of
simultaneously measuring and analyzing FSC,
SSC, and 3 spectral regions of fluorescence.
BD LSR: Expands the range of multicolor analysis
applications you can run on a benchtop cytometer, with
up to six fluorescence and two scatter parameters.
The LSR also provides high resolution DNA ploidy and
pulse processing technology that allows the
measurement of area, width and ratio of detector
pulses.
BD FACS Vantage SE: The FACS Vantage SE is an
analytical flow cytometer with sorting
capabilities. This instrument has 6-color
capability and can sort up to 20,000 cells/sec.
The FACS Vantage is equipped with high speed
and turbo sorting, as well as pulse processing
and automated single-cell deposition unit.
U OF C FLOW CYTOMETRY USER FACILITY
We offer individual training on
basic flow cytometry on an
BD FACScan (488 laser)
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
Future courses will include:
 Analysis of flow cytometric data
 FLOWJO
 Measurement of Apoptosis
 DNA and Cell Cycle Analysis
For more information please contact
Laurie Kennedy (210-8598)
(lkennedy@ucalgary.ca)
PROVIDING FLOW CYTOMETRY &
CELL SORTING SERVICES FEATURING
THE MOST ADVANCED FLOW
TECHNOLOGY
•Multi-color immunofluorescence
•DNA/Cell cycle analysis
•Intracellular cytokine measurements
•Fluorescence quantitation
•Multi-color, rare event and single cell sorting
•Comprehensive computer analysis
Flow Cytometry Core Facility, The University of Calgary
Rm 2580 - 3330 Hospital Drive NW, Calgary, AB T2N 4N1
tel. 403.220.7502
e-mail lkennedy@ucalgary.ca
www.flowlab.ucalgary.ca
PERSONNEL
Director: John D. Reynolds (reynolds@ucalgary.ca)
Operator: Laurie Robertson, HSC 2580
Tel: 220-7502 e-mail: robertl@ucalgary.ca
Operator and Instructor: Laurie Kennedy, HSC 2802
Tel: 210-8598 e-mail: lkennedy@ucalgary.ca
Please visit our website:
www.flowlab.ucalgary.ca