20140218_Immunology_Methodology_class

Report
Christine Zhang, PhD
Faculty of Medicine
University of Manitoba
Presentation Outline
 Basic Concept of Flow Cytometry analysis and instrumentation
 Applications of Flow Cytometry in Research Analysis
 Fluorescence-Activated Cell Sorting (FACS)
 Our Flow Cytometry Core Facility
Flow Cytometry Background
 A system that integrates electronics, fluidics, optics, laser technology and computer
analysis in a single platform.
 Allows for simultaneous multiparametric analysis of physical/chemical/biological
characteristics of cells or particles at the single-cell level by detecting fluorescence
intensity as they travel in suspension one by one past a sensing point
Emission light
Optical components
Detectors/amplifiers (PMT)
Excitation light
Lasers
Digital
(computer/software)
Flow chamber and
fluidics
Mixed cells with fluorescence label
Flow Cytometry Background
•
Cell or particle size; cytoplasmic granularity •
gene expression and transfer efficiency by
•
Cell surface antigens (immunophenotyping)
reporter like GFP
•
intracellular antigen (e.g. cytokine profiling) •
mRNA expression in living cells
•
Intracellular signalling (phospho-flow)
•
Protein-protein interaction by FRET
•
Cell viability/apoptosis
•
Activation sate and oxidative stress
•
Cell cycle, DNA content/synthesis
•
Stem cell side population analysis
•
Cell proliferation (BRDU and CFSE)
•
Cell sorting
•
Intracellular Ca2+ flux
•
Cell migration and adhesion
I am a cell…
I am a virus…
The Generation of Fluorescent Light
Jablonski Diagram of Fluorescence in 1935

Electron of a fluorochrome absorbs light energy at
certain wavelength (Ex)
 Excited to a higher energy level S1 from ground
level (S0).
 Stair-step vibrational relaxation in picosec
 Energy level returns to lower level and emit
fluorescent light at certain wavelength (Em)

Different fluorochromes have distinct Ex/Em
Variation in Ex/Em of Commencially Available Fluorophores
Excitation by lasers at
355nm laser (UV)
Emission Spectra of Alexa series
405nm laser (violet)
488nm laser (blue)
561nm laser (green)
633nm laser (red)
Each fluorochrome has its distinct excitation and emission spectra. Check if your
instrument can excite and detect your fluorochromes first
Flow Cytometry: How does it work
Step 1: Panel Design—find the best marker-fluorophore combo for your instrument
Your Marker Panel
Your Fluorophore Panel – fluorescence spectra viewer
T cells: CD3+, CD90.2+
• Effector T cell: CD4+, CD8+
• Treg: CD25+
B cells: CD19+, B220+
• Naïve B: CD27-CD138• Plasma B: CD27+CD138+
Mono/Mac: CD14+CD16- or CD16+
NK: NK1.1+,CD56+
Neutrophils: CD11chiCD11bhiCD16hi
mDCs: Lin-MHCIIhi CD11chiCD123pDCs: Lin-MHCII- CD11c-CD123+
•
minimize Spillover: Choose fluorochromes excited by different lasers, avoid fluorochromes in
adjacent channels
•
Be aware of the fluorochrome brightness index and marker expression: Bright
fluorochrome – low expression marker; Dim fluorochrome – high expression marker
Flow Cytometry: How does it work
Step 1: Example of the Marker-Fluorochrome Panel Design
Goal: characterize IL-10 and TGFβ expression in Tregs in human PBMC
Key: Choose colors for low expressing-intracellular proteins for which staining is complicated
first. Abundant easy-to-stain surface lineage markers last.
My Panel Design
Marker Panel
Fluorochrome Choice
CD4
BV510
CD25
PE-Cy7
Foxp3
APC
TGFβ
PE
IL-10
Alexa488
CD14
eFluo450
Fluorescence Parameters on FACSCanto
Laser
blue (488 nm)
Fluorochrome:
FITC, GFP, Alexa488
PE
PerCP, PerCP-Cy5.5
PE-Cy7
red (633 nm)
APC, Alexa647
APC-cy7
Violet (405 nm)
Pacific blue, Dapi, V450
AmCyan, V500, BV510
Take-Home Message: Know your marks (e.g. expression, surface or intracellular) and
fluorochromes (e.g. signal intensity, stability, etc.)
Flow Cytometry: How does it work (cont’d)
Step 2: Sample Preparation — fluorescence labeling proteins of your interest
GFP-vector transfected cells
Fluorescence-tagged Ab labeled cells
Anti-protein B Ab
Gene A
GFP
Gene B
RFP
Gene C
CFP
Anti-protein A Ab
Gene
D
YFP
Protein A
Protein B
Remember to optimize your staining protocol: e.g. cell detachment, collagenase
concentration, Antibody titration, staining buffer, suitable fix/perm solution, etc.
Flow Cytometry: How does it work (cont’d)
Step 2: Sample Preparation — fluorescence labeling proteins of your interest:
Cell Surface Staining
 Collect cells and resuspend in PBS + 1% Fetal Calf Serum/BSA
 Block FcγRs
 Staining cells with primary antibodies-fluorochrome
 Incubate on ice for 30 minutes to allow for antibody binding
 Wash, optional fixation step, resuspend, analyze
Intracellular staining/Phospho-Flow
 Collect Cells as above
 Block FcγRs
 Fix and permeabilize cells
 Rehydrate cells (preferred for phospho-flow analysis)
 Staining with primary antibodies-fluorochrome
 Incubate on ice for 30 minutes to allow for antibody binding
 Wash, resuspend, analyze
Flow Cytometry: How does it work (cont’d)
Step 2: Sample Preparation — Know Your Cells
Cell Preparation
• adherent cells need to trypsinize prior to staining  cell surface markers may be
affected
• large and sticky cells can be difficult to work with  clumping, clogging the flow cell
Non-specific Staining:
• FcR binding: can be blocked with antibody against FcR (Fc BLOCK)
• Dead or dying cells: bind every fluorescently labeled antibody at a high level
• Auto-fluorescence: some cells are intrinsically fluorescent without any staining at
different level.
Macrophage: High autofluorescence
DC: Low autofluorescence
Autofluorescence tends to increase in all cells after fixation and prolonged storage
Flow Cytometry: How does it work (cont’d)
Step 3: analysis on flow cytometer
FACSCalibur 2 laser system
(4 fluorescence channels)
LSRII 3 laser system (13 -15 fluorescence channels)
FACSCantoII 3 laser system
(8 fluorescence channels)
Inside the Flow Cytometer: Signal Generation
Laser Beam
Sheath Flow
Sample Flow
sheath tank
tube
sheath
filters/fluid
lines
Sample
injection tube
(SIT)
Collecting lens
Flow
Cell
interrogation
point
Flow chamber
Sheath
Sampl
e
Waste tank
1. Fluorescence-labeled Cells are carried to the
flow chamber by liquid stream
2. Florochromes are excited by laser light beam
Inside the Flow Cytometer: Signal Generation
3. Emitted fluorescent light pass through a set of optical filters
4. Light signals are amplified and detected by detectors (PMT).
5. Light signals are converted to digital signals and sent to
computer for analysis
LP filter
PMT
BP filter
Understanding the Optical Components: Longpass Filters
and Bandpass Filters
3) PMT
% of transmission
Longpass Filter
2) BP filter
Wavelength (nm)
1) LP filter
% of transmission
Bandpass FIlter
3) PMT
Wavelength (nm)
How to read a longpass filter and bandpass filter detection range
e.g.: 502LP  wavelength longer than 502nm will be allowed to pass through
e.g. 530/30 = 530nm +/- (30/2)nm = 530nm +/- 15nm = 515nm – 545nm
Flow Cytometry: Parameters
Forward Scatter: measures cell or particle size
Side Scatter: measures internal complexity or granularity of
the cell
Example of Fluorescence Parameters on LSR-II
3 Lasers, 16 parameters (Forward Scatter, Side Scatter, 14 fluorescent detectors)
Laser
blue (488 nm)
Mirror/Filter
505LP, 530/30
550LP, 575/25
600LP, 610/20
635LP, 670/14
735LP, 780/60
Fluorochrome
FITC, GFP, Alexa488
PE, dsRed, Alexa546, Cy3
PE-Texas Red, PI (live)
PE-Cy5.5, 7-AAD
PE-Cy7
red (633 nm)
660/20
710LP, 730/45
755LP, 780/60
APC, Alexa647, Cy5
Alexa700
APC-cy7, APC-Alexa750
Violet (405 nm)
450/40
475LP, 525/50
545LP, 560/20
575LP, 585/15
595LP, 605/12
630LP, 655/8
Pacific blue, Dapi, V450
AmCyan, CFP, V500
Qdot 565
Qdot 585
Qdot 605, BrilliantViolet605
Qdot 655
Flow Cytometry can detect cells or particles with a wide range of size
Size beads only (0.1 – 1.1μm)
Granularity (side scatter)
Viral particles
size (forward scatter)
Cell-derived microparticles
Primary mouse splenocytes
Flow Cytometry: Signal Conversion in PMT
1.
Laser
Voltage
Photon  Current  Voltage  Digital Signal
2.
Laser
Voltage
time
3.
Laser
Voltage
time
time
•
•
Fluorescent emissions are detected as a voltage pulse from photomultiplier tube (PMT)
detectors
The area, voltage and height of the voltage pulse is measured
Flow Cytometry: Data Analysis
Flow cytometry data can be plotted in several different ways:
•
the axes of the graphs represent fluorescence intensity data, usually plotted on a log
scale
• for histograms, the y axis is cell number
Histograms
Flow Cytometry Analysis – from Simple to Complex
Monoclonal stable cell line
More
Granular
Side Scatter (SSC)
Dead Cells
and debris
Bigger Cells
smaller cells
Forward Light Scatter (FSC)
Live
Cells
Bigger
GFP expression
Whole blood
100
80
% of M a x
GFP+: 87.5%
60
SSC
cell count
40
Receptor A
20
Receptor B
0
10
FSC
0
1
2
10
10
FL1-H: receptor anti-myc
GFP (MFI)
10
3
10
4
Brighter
Flow Cytometry Analysis – from Simple to Complex
Single PE Positive
Population
Double Positive
Population
CD8-PE
Negative
Population
CD4-FITC
Single FITC Positive
Population
Wood B. 2006. Arch Pathol Lab Med. 130:680–690
Be Aware of signal spill-over for large panel analysis
•
•
Fluorescence signal spill over = signal from one fluorochrome (e.g. FITC) being picked
up by detector for another fluorochrome (e.g. PE).
For compensation, use single stained controls for every fluorochrome you use along
with unstained control
FITC only
Before compensation
unstained
Compensation to
remove signal spillover
after compensation
PE - %FITC  take out x% of the signal
in PE that is due to spill-over from FITC
Signal Spillover and Color Compensation
Tips on compensation:
• Compensation is specific for fluorochromes, NOT for cell types  compensation values are valid
for all cell types
• Try Compensation beads if your cell samples are precious or if your marker expression is low.
• Choose good markers that gives sharp clear positive peak away from negative peak
 lymphocytes: CD4, CD8, CD90, CD19. DC/neutrophils: Gr-1, CD11b)
• Expression should be equal or higher than the experimental samples
• Minimize spillover by spreading your colors of choice over different lasers and avoid adjacent
channels
• Manual compensation to double check
uncompensate
d
compensated
Median values both = ~3.2
under compensated
over compensated
Western Blotting
Fluorescence
Microscopy
Flow Cytometry
Pros
• semi-quantitative
• no specialized
instrument detect
• Detect different
forms of a single
molecule
• Qualitative
• Single cell based
• Show
molecular/cellular
expression and
localization
• fixed and live
•
•
•
•
•
•
Cons
• Not single cell based
• not very sensitive
• protein denatured
• not quantitative
• only detect total or surface
protein level
• protein subcellular
localization; cellular
location
• protein colocalization
• inter-/intra-molecular
interaction
• live cell imaging
• surface/total expression
level of proteins.
• developmental biology
study using multiple lineage
markers
Applications • protein expression,
maturation,
phosphorylation,
ubiquitylation,
localization by
fractionation.
Quantitative
Sensitive
No photobleaching
Single cell based
fixed and live
expression of multiple
molecules on the same cell
• And more…
Presentation Outline
 Basic Concept of Flow Cytometry analysis and instrumentation
 Applications of Flow Cytometry in Research Analysis
 Fluorescence-Activated Cell Sorting (FACS)
 Our Flow Cytometry Core Facility
Flow Cytometry Application
Workflow
Maecker H. Nature Reviews Immunology 12, 191-200
Marker Panel
T cells: CD3+, CD90.2+
• Effector T cell: CD4+, CD8+
• Treg: CD25+
B cells: CD19+, B220+
• Naïve B: CD27-CD138• Plasma B: CD27+CD138+
NK: NK1.1+,CD56+
Mono/Mac: Lin-CD14+
• Classical: CD16• Non-classical: CD16+
mDCs: Lin-HLA-DRhi CD11chiCD123pDCs: Lin-HLA-DR- CD11c-CD123+
Cytokine profiling on the desired population:
e.g. IFNα, IL10, CXCR3, CCR5, TFNα
Maecker H. Nature Reviews Immunology 12, 191-200
10-color analysis on moncytes subsets (viability marker, lineage markers and cytokines all in 1 tube)
PMBCs
Exclude doublets
Exclude T, B, NK
CCR2
CD16
CXCR3
Exclude the dead
TNFα
Cytokine profiling
IL10
CD14
3 Monocyte subsets
Exclude HLA-DR neg
•
•
•
B-cell neoplasia (e.g. CLL) can be diagnosed by flow cytometry, WBC count and clinical history.
more than 5x109 monoclonal B cells in the blood  CLL
Elevated B cell number alone is not enough to diagnose various subclasses of B-Cell Neoplasia
>40% CD19+ cells in PBMC
CD19
CD23
Chronic Lymphocyte
Leukemia (CLL)
CD19+CD5+CD23+
CD19
CD11c
CD5
CD23
CD5
CD5
CD5
CD19
CD5
Mantle Lymphocyte
Leukemia (MCL)
CD19+CD5+CD23-
CD19
CD20
Hairy Cell Leukemia (HCL)
CD19+CD5-CD20+CD11c+
Maryalice Stetler-Stevenson, Flow Cytometry Unit, NIH
Flow Cytometry Application
Cell Surface Staining
 Collect cells
 Block FcγRs
 Staining cells with primary antibodies-fluorochrome
 Wash, resuspend, analyze
Intracellular staining/Phospho-Flow
 Collect Cells
 Block FcγRs
 Fix and permealize cells
 Rehydrate cells (preferred for phospho-flow analysis)
 Staining with primary antibodies-fluorochrome
 Wash, resuspend, analyze
Key Ingredient: good phospho-specific antibodies directly conjugated to fluorophore
Note: phosflow is limited only to high abundance protein that are highly phosphorylated on
very unique sites that can be detected with specific antibodies (a small subset of signaling
molecules)
Krutzik P. 2011. Flow Cytometry Protocol; Chapter 9, Fig. 2
Flow Cytometry Application
Cell Cycle Phases
DNA histogram
G0/G1 phase (2N)
S phase (2 - 4N)
Debris
G2/M phase (4N)
Common dyes that bind stoichimetrically to DNA: Propidium Iodide, Hoechst
33342, DAPI, 7AAD ,DRAQ5, etc
Tabll A. 2011. Liver Biopsy; Chapter 7, Fig. 2
Normal cell (diploid)
G0/G1 = 60%
Diploid
S = 13%
G2/M = 27%
tetraploid
Tumor cell (aneuploidy)
G0/G1 = 79.5%
S = 12.7%
hyperdiploid
G0/G1
Diploid
Tumor
G2/M = 7.8%
Count
Count
hypertetraploid
DNA CONTENT
G2/M
S
DNA CONTENT
Ross J. 2003. Am J Clin Pathol. 120: S72-S84
Flow Cytometry Application
Apoptosis
Cell dehydration
Membrane blabbing
Chromatin condensation
Apoptotic bodies
Loss of PS asymmetry
Nucleus collapse
Propidium iodide
31% later stage
33% Viable
35% early stage
Annexin V (phosphatidyl serine)
•
•
Early and late apoptosis (programmed cell death) can be measured based on several
different type of cellular alterations
Each type of alteration can be detected by flow
1) Activation of caspases: detected by staining with antibodies detecting cleaved caspases
or caspase substrates OR staining with fluorescently-labeled caspase inhibitors (Casp-Glow)
2) Mitochondrial dysfunction: detect changes in membrane potential with Rhodamine 123,
TMRE, MitoTracker dyes or cytochrome C release using specific antibodies
3) Alterations in membrane symmetry: phosphatidyl serine translocates from cytoplasmic
to extracellular side of membrane > detected by annexin V binding (note: membrane
inversion also occurs during granule release in neutrophils, mast cells, etc)
4) Loss of membrane integrity: apoptotic cells become permeant to DNA-binding
dyes such as DAPI or PI
5) DNA fragmentation: TUNEL assay > TdT enzymatic incorporation of fluorescent
nucleotide analogues
Flow Cytometry Application
BrdU
count
BrdU/EdU: incorporated into the newly synthesized DNA of replicating cells (during the S
phase)
CSFE: measure cell division as CSFE fluorescence intensity is halved within daughter cells after
each cell division
EdU
low-proliferating BM cells
Highly-Proliferating atrial cells
Propidium Iodide
Non-proliferating cells
Proliferating cells
Presentation Outline
 Basic Concept of Flow Cytometry analysis and instrumentation
 Applications of Flow Cytometry in Research Analysis
 Fluorescence-Activated Cell Sorting (FACS)
 Our Flow Cytometry Core Facility
FACSAriaIII 3 laser system (15-16
fluorescence channels)
Fluorescence-Activated Cell Sorting (FACS)
FACS: a specialized type of flow cytometry to sort a
heterogeneous mixture of cell suspension
Mixture of cells
to be sorted
Features
• Sort up to 4 populations of interests
laser
PMT
•
15 fluorescence color simultaneous on the same cell
•
Sort different types cells
o
o
o
o
nozzle
New drop
+
empty drop
_
_
_
_
_
_
_
_
+
+
+
_
_
•
+
+
+
+
+
+
_
•
v
Multi-purpose sorting
o
o
o
o
o
+
+
Primary BM, PBMC, mouse splenocytes
Any types of cell lines
Large fragile cells like activated neutrophiles, lung DCs
Sticky and hard to sort cells (e.g. solid tumor cells,
neuron cells)
7ml round bottom tube, 15ml conical tubes.
Tissue culture plates, 96 well PCR plates
Microscope slides including multiwell chamber slides
Single cell sorting
Different modes to maximize sort purity (99% for qPCR)
or recovery (for assays requiring large number of cells
sterile sorting, sample agitation, temperature control
FACS cell soring pros and cons
Mixture of cells
to be sorted
laser
FACS sorting
Pros:
• Good for sorting very rare population or any
populations of interests.
• Accommodate large panel of markers (up to 15
colors)
• Sort up 4 populations simultaneously
• Cell sample quality check by flow cytometry
before sorting.
• High purity, low death rate after sorting
PMT
nozzle
New drop
+
empty drop
_
_
_
_
_
_
_
_
+
+
+
_
_
+
+
+
+
+
+
+
+
_
v
Cons:
• Require specialized instrument and dedicated
personnel
• May take longer to sort a large number of cells
Sorting Example 1: Enrichment of Ramos Cells transiently transfected
with 4 different constructs
Before Sorting
EGFP
SHIP
PD
Count
After Sorting
GFP
Y944F
Sorting Example 2: sort mature and immature neutrophils from mouse BM
SSC
Before Sorting
CD11b
FSC
Gr-1
im neu
After Sorting
ma neu
BD FACSCanto-II Digital Flow Cytometry Analyzer
BD LSR-II Digital Flow Cytometry Analyzer
Location: Room 466, Apotex Center, University of Manitoba
Bannatyne Campus
Location: Room 536, Basic Medical Science Building,
University of Manitoba Bannatyne Campus
3 Lasers: 1) 488 nm blue laser; 2) 633 nm red laser; 3) 405
nm violet laser
3 Lasers: 1) 488 nm blue laser; 2) 633 nm red laser; 3) 405
nm violet laser
10 parameters: FSC, SSC, 4 fluorescent detectors off 488 nm,
2 fluorescent detectors off 633 nm, 2 fluorescent
parameters off 405 nm.
16 parameters: FSC, SSC, 5 fluorescent detectors off 488 nm,
3 fluorescent detectors off 633 nm, 6 fluorescent
parameters off 405 nm
BD FACSAriaIII Digital Cell Sorter
Location: Room 462, Apotex Center, University of Manitoba Bannatyne Campus.
3 Lasers: 1) 488 nm blue laser; 2) 633 nm red laser; 3) 405 nm violet laser
17 parameters: Forward Scatter, Side Scatter, 6 fluorescent detectors off 488 nm, 3 fluorescent detectors off 633 nm, 6
fluorescent parameters off 405 nm
Features: sample agitation; temperature control; two-way or four-way sorting into tubes, multiwell plates or microscope
slides; Aerosol Management Option (AMO); housed in a ClassII Type A2 biosafety cabinet; accommodate most cell types.
Christine Zhang, Ph.D.
Flow Cytometry Core Facility Manager
Faculty of Medicine, University of Manitoba
413 Apotex Center, 750 McDermot Ave.
Tel: (204) 294-0691
Email: [email protected]
http://umanitobaflow.ca/
http://www.cyto.purdue.edu/flowcyt/educate.htm
http://www.lifetechnologies.com/ca/en/home/life-science/cell-analysis/flowcytometry/flow-cytometry-technical-resources.html
http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/

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