What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry...

46
What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry Rui Gardner IGC – April 29, 2010

Transcript of What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry...

Page 1: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

What is Flow Cytometry?

Introduction to Flow CytometryIGC Workshop

Applications in Flow Cytometry

Rui GardnerIGC – April 29, 2010

Page 2: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – 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

Page 3: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Cell Phenotyping

Page 4: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Immunophenotyping

CD3+ CD3+ CD4+CD3+ CD4+ CD25-

CD3+ CD4+ CD25+

CD3+

CD4+ CD25- CD25+

Page 5: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Cell Activation

Page 6: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Activation

Activation

IL-7

67%

Medium

23%

FSC x SSC – Cell size

Page 7: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Activation

Activation markers: CD69, CD71, etc

Page 8: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Cell Cycle

G2

M

G1

S

G0

Page 9: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Cell Cycle

DNA content analysis - Propidium Iodide (PI)

G2

M

G1

S

G0

G0/G1

S-phase

G2/M

Fluorescence (DNA content)

Page 10: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Cell Cycle Analysis

Cell Cycle Analysis Software

G0/G1

SG2/M

Fluorescence Intensity

Cell

Num

ber

Page 11: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

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

Page 12: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

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

Page 13: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Apoptosis

Page 14: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

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

Page 15: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Apoptosis

Propidium Iodide (fixed cells)

DNA degradationDNA Degradation

Page 16: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Apoptosis

Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells)

Page 17: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Apoptosis

Annexin V plus propidium Iodide

Page 18: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Apoptosis (intracellular staining)

Fix and permeabilize Add

Antibody

Analyse by Flow

Cytometry

Page 19: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Apoptosis – Bcl-2 family members

Page 20: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

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

Page 21: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Cell Proliferation

Page 22: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Tracking Cell Proliferation with CFSE

STAIN WITH CFSE

Dilution of CFSE

Cell Divisions

CELL

Page 23: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Tracking Cell Proliferation with CFSE

IL-7 IL-7+ DMSO

IL-7+ PI3K Inhibitor IL-7+ Erk Inhibitor

Page 24: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Activation of Signaling Pathways

Page 25: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Activation of signalling pathways

Phospho-protein detection

Page 26: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Activation of signalling pathways

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Activation of signalling pathways

Discrimination of High vs. Low responders

pStat1

pStat1

Page 28: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Activation of signalling pathways

Discrimination of simultaneous vs. non-simultaneous activation of different pathways in single cells

Page 29: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Combining Surface Markers with Phospho-staining

Page 30: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Cytokine Secretion

Page 31: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Multiplex Bead Arrays

bead coated with capture antibody for particular cytokine

Cyto

kine

s

Amount Cytokine

Page 32: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Multiplex Bead Arrays

NEAT 1/8

1/64 NEG

Page 33: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Calcium Flux

Page 34: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

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

Page 35: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Telomere Length

Page 36: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Telomere Length

Page 37: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Today’s Future Applications

Page 38: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Amnis Image Stream

Page 39: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Amnis Image Stream

Page 40: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

What is Flow Cytometry?

Introduction to Flow CytometryIGC Workshop

Applications in Flow Cytometry (end)

Rui GardnerIGC – April 29, 2010

Page 41: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Sorting Applications

Page 42: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

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.

Page 43: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

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)

Page 44: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

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

Page 45: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

Establishment of Cell Clones

Sort single cell into each well

time

Clone A

Clone B

Clone C

Page 46: What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010.

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