What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Applications in Flow Cytometry
IGC – April 29, 2010
Outline
Potential Applications of Flow Cytometry
Cell State
Cell Function
• Immunophenotyping
• Cytokine Secretion
• Cell activation
• Activation of signalling pathways
• Cell cycle
• Calcium flux
• Cell proliferation
• Levels of intracellular reactive oxygen species
• Apoptosis
• Telomere length
• Differentiation
• Identification of “stem cells”
Cell Separation
• Sorting
Cell Phenotyping
Immunophenotyping
CD3+
CD4+
CD25-
CD25+
CD3+ CD4+ CD25CD3+
CD3+ CD4+
CD3+ CD4+ CD25+
Cell Activation
Activation
FSC x SSC – Cell size
Medium
IL-7
23%
67%
Activation
Activation
Activation markers: CD69, CD71, etc
Cell Cycle
M
G0
G1
G2
S
Cell Cycle
DNA content analysis - Propidium Iodide (PI)
M
G0/G1
G0
G1
G2/M
G2
S
S-phase
Fluorescence (DNA content)
Cell Cycle Analysis
Cell Cycle Analysis Software
Cell Number
G0/G1
S
G2/M
Fluorescence Intensity
Cell Cycle - Bromodeoxyuridine (BrdU) method
Propidium Iodide plus BrdU staining
• Thymidine analog
104
• Taken up by cells in S-phase
S Phase
• Usually in combination with Propidium iodide
103
102
101
G1
0
G2/M
20 0
60 0
40 0
FL 3-H
Propidium Iodide
New Click-It DNA technology from Invitrogen does not require DNA denaturation.
80 0
10 00
Cell Cycle - G0/G1 discrimination
Cell Count
G0/G1
RNA Content
Pyronin Y plus Hoechst 33342/33258
G0
S
G2/M
G1
S
G2/M
Apoptosis
Cell Death
CELL DEATH – FSC x SSC
0
10
0
S S C-H : S SC -H eight ( Log S cal e)
Activation
10
10
10
10
10
200
400
600
FSC-H: FS C-Height
800
3
10
2
10
1
10
37.4%
37.4
0
10
400
600
800
0
1000
200
400
600
FSC-H: FS C-Height
800
2
1
17.1%
17.1
0
10
10
10
10
0
200
400
600
800
1000
10
1
10
26.2%
26.2
0
10
0
10
3
2
1000
4
10
200
400
600
FSC-H: FS C-Height
800
3
2
1
10
2
10
1
10
26.1%
26.1
0
10
0
200
400
600
800
1000
10
10
10
10
0
10
3
25.6%25.6
0
1000
4
Viability
10
10
0
10
200
15.9
15.9%
1000
4
0
1
3
10
4
S S C-H : S SC -H eight ( Log S cal e)
10
27.6
27.6%
10
10
200
400
600
FSC-H: FS C-Height
800
3
2
28.8
28.8%
10
10
10
0
10
200
400
600
2
1
0
200
FS
10
0
3
0
4
1
4
1000
S S C-H : S SC -H eight ( Log S cal e)
1
2
10
4
S S C-H : S SC -H eight ( Log S cal e)
10
3
S S C-H : S SC -H eight ( Log S cal e)
2
10
Me
100nM Rapa
S S C-H : S SC -H eight ( Log S cal e)
10
10
Medium
S S C-H : S SC -H eight ( Log S cal e)
10
3
4
Viability
10
10
S S C-H : S SC -H eight ( Log S cal e)
10
4
S S C-H : S SC -H eight ( Log S cal e)
S S C-H : S SC -H eight ( Log S cal e)
Viability
10
100nM Rapa
Activation
Medium
Thymocytes
Activation
T-ALL
800
1000
4
3
2
1
0
0
200
Apoptosis
Propidium Iodide (fixed cells)
DNA
degradation
DNA Degradation
Apoptosis
Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells)
Apoptosis
Annexin V plus propidium Iodide
Apoptosis (intracellular staining)
Fix and
permeabilize
Analyse by
Flow
Cytometry
Add
Antibody
Apoptosis – Bcl-2 family members
Apoptosis – Activated forms of Caspases
Untreated
Etoposide
Flow cytometric analysis of Jurkat cells, untreated (blue) or
etoposide-treated (green), using Cleaved Caspase-3 (Asp175)
Antibody (Alexa Fluor® 488 Conjugate).
Cell Proliferation
Tracking Cell Proliferation with CFSE
Dilution of CFSE
STAIN WITH
CFSE
CELL
Cell Divisions
Tracking Cell Proliferation with CFSE
IL-7
IL-7+ PI3K Inhibitor
IL-7+ DMSO
IL-7+ Erk Inhibitor
Activation of
Signaling Pathways
Activation of signalling pathways
Phospho-protein detection
Activation of signalling pathways
Activation of signalling pathways
Discrimination of High vs. Low responders
pStat1
pStat1
Activation of signalling pathways
Discrimination of simultaneous vs. non-simultaneous
activation of different pathways in single cells
Combining Surface Markers with Phospho-staining
Cytokine Secretion
Multiplex Bead Arrays
Cytokines
bead coated with capture antibody
for particular cytokine
Amount Cytokine
Multiplex Bead Arrays
NEAT
1/8
1/64
NEG
Calcium Flux
Calcium Flux
Effects of T cell receptor stimulation on CD4 cell
ionized calcium concentration ([Ca2+]i).
Fluorescence-imaging of human erythrocytes
treated with PGE2 using the calcium
fluorophor Fluo-4
Telomere Length
Telomere Length
Today’s
Future Applications
Amnis Image Stream
Amnis Image Stream
What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Applications in Flow Cytometry (end)
IGC – April 29, 2010
Sorting Applications
Sorting Immunophenotipic populations
CD3+
CD4+
CD25-
CD25+
CD3+ CD4+ CD25-
CD3+
Transcriptomics (RNA)
Genomics (DNA)
Metabolomics (metabolites)
CD3+ CD4+
CD3+ CD4+ CD25+
Fluorescence microscopy
FISH
Functional Studies
Etc.
Establishing Fluorescent Cell Lines
Carina Santos (IMM)
Interphase
Cultured
mCherry signal
Anaphase
Human hepatoma cell line
Expressing α-tubulin fused with mCherry
mCherry signal
mCherry signal
Chromosome sorting
Human cell line with translocation between
chromosome 2 and chromosome 17
AT-rich DNA signal
Normal human cell line
GC-rich DNA signal
Establishment of Cell Clones
Clone A
Clone B
Clone C
Sort single cell into each well
time
Future Advances
• More colours for immunofluorescence
(quantum dots, tandem dyes)
• Reduced laser size and capillary flow
techniques mean smaller instruments
• Instruments can now image cell at point of
laser interrogation