What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry...
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Transcript of What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry...
What is Flow Cytometry?
Introduction to Flow CytometryIGC Workshop
Applications in Flow Cytometry
Rui GardnerIGC – April 29, 2010
Outline
Potential Applications of Flow Cytometry
• Immunophenotyping
• Cell activation
• Cell cycle
• Cell proliferation
• Apoptosis
• Differentiation
• Identification of “stem cells”
• Cytokine Secretion
• Activation of signalling pathways
• Calcium flux
• Levels of intracellular reactive oxygen species
• Telomere length
• Sorting
Cell State Cell Function
Cell Separation
Cell Phenotyping
Immunophenotyping
CD3+ CD3+ CD4+CD3+ CD4+ CD25-
CD3+ CD4+ CD25+
CD3+
CD4+ CD25- CD25+
Cell Activation
Activation
Activation
IL-7
67%
Medium
23%
FSC x SSC – Cell size
Activation
Activation markers: CD69, CD71, etc
Cell Cycle
G2
M
G1
S
G0
Cell Cycle
DNA content analysis - Propidium Iodide (PI)
G2
M
G1
S
G0
G0/G1
S-phase
G2/M
Fluorescence (DNA content)
Cell Cycle Analysis
Cell Cycle Analysis Software
G0/G1
SG2/M
Fluorescence Intensity
Cell
Num
ber
Cell Cycle - Bromodeoxyuridine (BrdU) method
Propidium Iodide plus BrdU staining
• Thymidine analog
• Taken up by cells in S-phase
• Usually in combination with Propidium iodide
New Click-It DNA technology from Invitrogen does not require DNA denaturation.
0 200 400 600 800 1000FL3-H
Anti -
B rdU
FI T
C
G1 G2/M
S Phase
101
102
103
104
Propidium Iodide
Cell Cycle - G0/G1 discrimination
Pyronin Y plus Hoechst 33342/33258
G0/G1
S
G2/M
G0
S
G2/M
G1
Cell
Coun
tRN
A Co
nten
t
Apoptosis
Cell Death
CELL DEATH – FSC x SSC
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SS
C-H
: SS
C-H
eigh
t (Lo
g S
cale
)
27.6
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SS
C-H
: SS
C-H
eigh
t (Lo
g S
cale
)
37.4
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SS
C-H
: SS
C-H
eigh
t (Lo
g S
cale
)
15.9
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SS
C-H
: SS
C-H
eigh
t (Lo
g S
cale
)
17.1
Via
bilit
yA
ctiv
atio
n
Medium 100nM Rapa
27.6% 15.9%
37.4% 17.1%
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SS
C-H
: SS
C-H
eigh
t (Lo
g S
cale
)
26.2
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SS
C-H
: SS
C-H
eigh
t (Lo
g S
cale
)
26.1
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SS
C-H
: SS
C-H
eigh
t (Lo
g S
cale
)
28.8
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SS
C-H
: SS
C-H
eigh
t (Lo
g S
cale
)
25.6
Via
bilit
yA
ctiv
atio
n
Medium 100nM Rapa
26.2% 25.6%
26.1% 28.8%
T-ALL Thymocytes PBMCs
Apoptosis
Propidium Iodide (fixed cells)
DNA degradationDNA Degradation
Apoptosis
Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells)
Apoptosis
Annexin V plus propidium Iodide
Apoptosis (intracellular staining)
Fix and permeabilize Add
Antibody
Analyse by Flow
Cytometry
Apoptosis – Bcl-2 family members
Apoptosis – Activated forms of Caspases
Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate).
UntreatedEtoposide
Cell Proliferation
Tracking Cell Proliferation with CFSE
STAIN WITH CFSE
Dilution of CFSE
Cell Divisions
CELL
Tracking Cell Proliferation with CFSE
IL-7 IL-7+ DMSO
IL-7+ PI3K Inhibitor 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
bead coated with capture antibody for particular cytokine
Cyto
kine
s
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 CytometryIGC Workshop
Applications in Flow Cytometry (end)
Rui GardnerIGC – April 29, 2010
Sorting Applications
Sorting Immunophenotipic populations
Transcriptomics (RNA)Genomics (DNA)Metabolomics (metabolites)
CD3+ CD3+ CD4+CD3+ CD4+ CD25-
CD3+ CD4+ CD25+
CD3+
CD4+ CD25- CD25+
Fluorescence microscopyFISHFunctional Studies
Etc.
Establishing Fluorescent Cell Lines
Interphase
Anaphase
Human hepatoma cell lineExpressing α-tubulin fused with mCherry
mCherry signal
mCherry signal mCherry signal
Sorted
CulturedCarina Santos (IMM)
Chromosome sorting
Human cell line with translocation between chromosome 2 and chromosome 17
Normal human cell line
GC-rich DNA signal
AT-r
ich
DN
A si
gnal
Establishment of Cell Clones
Sort single cell into each well
time
Clone A
Clone B
Clone C
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