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