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What is Flow Cytometry ?. Introduction to Flow Cytometry. IGC Workshop. Applications in Flow Cytometry. Rui Gardner. IGC – April 29, 2010. Outline. Potential Applications of Flow Cytometry. Cell State. Cell Function. Immunophenotyping Cell activation Cell cycle - PowerPoint PPT Presentation
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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 -
Brd U
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
SSC
-H: S
SC-H
eigh
t (Lo
g Sc
ale)
27.6
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SSC
-H: S
SC-H
eigh
t (Lo
g Sc
ale)
37.4
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SSC
-H: S
SC-H
eigh
t (Lo
g Sc
ale)
15.9
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SSC
-H: S
SC-H
eigh
t (Lo
g Sc
ale)
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
SSC
-H: S
SC-H
eigh
t (Lo
g Sc
ale)
26.2
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SSC
-H: S
SC-H
eigh
t (Lo
g Sc
ale)
26.1
0 200 400 600 800 1000FSC-H: FSC-Height
10 0
10 1
10 2
10 3
10 4
SSC
-H: S
SC-H
eigh
t (Lo
g Sc
ale)
28.8
0 200 400 600 800 1000FSC-H: FSC-Height
100
101
102
103
104
SSC
-H: S
SC
-Hei
ght (
Log
Scal
e)
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
DNA
signa
l
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