FLOW CYTOMETRY - korambiotech.comkorambiotech.com/upload/bbs/2/Cytometry.pdfTo this end, flow cytometry has been widely applied to the semi- quantitative analysis of mixed populations

P P

P

P

P

PP

P

PP

PP

Tyr505

TCR

Tyr319

Tyr171

Ras-Erk

Rho/Racpathways

CD4

LckTyr394

Grb2

PLC 1γ

Tyr142

Tyr191

Tyr783

Tyr113

Tyr128

LAT

SLP76 Vav

PIP2

DAGIP3

PKCCa2+

Calcineurin

ZAP-70Tyr493

Lymphocyte/Leukemia Signaling

MAPK Signaling and Proliferation

Survival/Apoptosis

Jak/Stat Signaling

Cell Cycle

Receptor Tyrosine Kinases

FLOW CYTOMETRYand Intracellular Signaling

T he study of cell signaling and human disease relies on the use of the most powerful

techniques and reagents. To this end, flow cytometry has been widely applied to the semi-

quantitative analysis of mixed populations of cells through the use of multiple surface

markers. The addition of DNA staining allows for further characterization of cell cycle-specific pro-

tein expression. Recent use of intracellular staining with activation-state-specific antibodies

allows for sensitive, rapid analysis of signal transduction in mixed populations of cells.

For example, cell cycle analysis may now include determination of the activation states of regula-

tory proteins such as Rb, CDKs and cyclins. The study of diseases such as leukemia or lymphoma

may be performed on well characterized patient samples in which the expression and activation

states of signaling proteins, such as the STAT proteins, receptor tyrosine kinases and downstream

targets, are directly analyzed. These new approaches to determining the mechanisms driving normal

and disease processes in biological systems take advantage of the power of flow cytometry in

addressing the needs of the researcher and clinician.

2

visit www.cellsignal.com for more product and reference information

Even

ts

Zap-70 (Alexa Fluor® 647)

A

CD3

(PE)

103

102

101

100

100 101 102 103


100 101 102

103 104

50

100

150

200


100 101 102 103 104

50

100

140


A11.3%

A366.7%

A421.6%

103

102

101

100

100

101

102

103

A210.5%

A110.5%

A32.3%

A40.5%

103

102

101

100

100

101

102

103

A286.8%

Phospho-Zap-70 (Tyr319) (FITC)

Phos

pho-

p44/

42 M

APK

(PE)

Flow cytometric analysis of Jurkat cells, untreated (left) or anti-CD3 activated (right), usingPhospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody and Phospho-p44/42 MAPK (Thr202/Tyr204)(E10) Mouse mAb #9106. Anti-CD3 activation increases the intensity of label with both anti-bodies.

Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody #2701

B

B

A

A. Flow cytometric analysis of Ramos B cells (blue) and Jurkat T cells (green), usingZap-70 (136F12) Rabbit mAb (Alexa Fluor® 647) Conjugate.

B. Flow cytometric analysis of human peripheral blood lymphocytes, using Zap-70(136F12) Rabbit mAb (Alexa Fluor® 647 Conjugate). High Zap-70 stain was observedin CD3-positive T cells (green), while low Zap-70 stain was observed in CD3-nega-tive B cells (red).

Tyr505

TCR

Tyr319

Tyr171

Ras-Erk

Rho/Racpathways

CD4

LckTyr394

Grb2

PLC 1γ

Tyr142

Tyr191

Tyr783

Tyr113Tyr128

LAT

SLP76 Vav

PIP2

DAGIP3

PKCCa2+

Calcineurin

ZAP-70Tyr493

ZAP-70: CLL Prognostic Marker

Flow cytometric analysis of two different CLL patient blood samples showing lowZap-70 in one (A) and higher Zap-70 in another (B), using Zap-70 (136F12) RabbitmAb (Alexa Fluor® 647 Conjugate). Results courtesy of Esoterix Center forInnovation. red=CLL cells, blue=T cells, green=NK cells, grey=ungated.

It has recently been shown that ZAP-70 expression is correlatedwith disease progression and survival in patients with chronic lym-phocytic leukemia (CLL). ZAP-70, a Syk family protein tyrosinekinase, plays a critical role in mediating T cell activation in responseto T cell receptor (TCR) engagement. Tyrosine phosphorylation ofZAP-70 correlates with increased kinase activity and downstreamsignaling events leading to the activation of the PLC-γ1-dependentand Ras-dependent signaling cascades in antigen-stimulated T cells.The use of flow cytometry for determining ZAP-70 expression in CLLpatient samples allows for the simultaneous detection of ZAP-70expression in additional hematopoietic cell types including T cells, Bcells and NK cells. The ZAP-70 expression in these other cell typesprovides internal positive and negative references for determiningZAP-70 expression in the CLL cells.

Zap-70 (136F12) Rabbit mAb (Alexa Fluor® 647 Conjugate) #2707

Lymphocyte/Leukemia Signaling

Even

ts

Phospho-FLT3 (Tyr591) Alexa Fluor® 488

3

■ Orders 1 877 616-CELL [email protected] ■ Technical Support 1 877 678-TECH [email protected]

Sos

Ras

MAPK

PI3K Gab2

Cbl CrkL HCK

AKT

CAS paxillin

Stat5

Survival

Proliferation

Cell movement

Proliferation, Survival

Bcl-XL,Cyclins, c-myc, Mcl-1

Grb2

Tyr177 Tyr245 Tyr412 Thr735

Bcr Abl

Lymphocyte/Leukemia SignalingCML Biomarkers: Monitor Gleevec® Response

AML: FLT3 Signaling

It has been demonstrated that the Bcr-Abl oncoprotein, a protein tyro-sine kinase, is the causative agent in human chronic myelogenousleukemia (CML). The Bcr-Abl fusion results in production of a constitu-tively active tyrosine kinase, which causes the autophosphorylation ofBcr-Abl and the phosphorylation of downstream substrates. In CML, Bcr-Abl phosphorylates CrkL, an adaptor protein involved in cell adhe-sion, migration and immune response. Similarly, transformation with Bcr-Abl causes constitutive Tyr694 phosphorylation and activation of Stat5.Recently, Gleevec® (STI-571), a small molecule inhibitor of the Abl kinase,has been approved for the treatment of CML. However, resistance toGleevec® has been observed in CML patients, leading to the developmentof new targeted drugs for use in treating CML patients. Signal transduc-tion analysis of CML using flow cytometry and activation-specific anti-bodies will aid in the development and use of Gleevec® and new targetedtherapies to CML.

FLT3 is a receptor tyrosine kinase expressed by hematopoieticprogenitor cells in the bone marrow, thymus and lymph nodes.Amplification, mutation or overexpression of FLT3 results inaberrant downstream signaling thus promoting cell survival,proliferation and inhibition of apoptosis. High expression of FLT3is observed in 70% of acute myelogenous leukemia (AML) cases,B-precursor cell acute lymphoblastic leukemia (ALL), a fractionof T-cell ALL and CML in lymphoid blast crisis. Most commonmutations of FLT3 kinase in cases with AML are internal tandemduplications and activation loop mutations. These mutationsresult in constitutive FLT3 activity that causes increased andpersistent downstream signaling, primarily through the MAPK,Jak/Stat and Akt pathways.

Phospho-c-Abl (Tyr245)

Even

ts

Flow cytometric analysis of K562 cells,untreated (green) or STI-571-treated(blue), using Phospho-c-Abl (Tyr245)Antibody compared to a nonspecificnegative control antibody (red).

Phospho-Bcr (Tyr177)

Even

ts

Flow cytometric analysis of K562 cells,untreated (green) or STI-571-treated (blue),using Phospho-Bcr (Tyr177) Antibodycompared to a nonspecific negative controlantibody (red).

Phospho-c-Abl (Tyr245) Antibody #2861

Phospho-Bcr (Tyr177) Antibody #3901

Phospho-CrkL (Tyr207)

Even

ts

Flow cytometric analysis of K562 cellsuntreated (blue) or STI-571-treated(green), using Phospho-CrkL (Tyr207)Antibody compared to a nonspecificnegative control antibody (red).

Phospho-CrkL (Tyr207) Antibody #3181

Flow cytometric analysis of K562cells, using Phospho-Stat5 (Tyr694)(14H2) Mouse mAb untreated (blue)or STI-571-treated (green).

Phospho-Stat5 (Tyr694) (14H2) Mouse mAb #9356

Phospho-FLT3 (Tyr591) Antibody (Alexa Fluor® 488 Conjugate) #3459

Phospho-Stat5 (Tyr694)

Even

ts

Flow cytometric analysis of SEM cells, treat-ed with a FLT3 inhibitor (blue) or untreated(green), using Phospho-FLT3 (Tyr591)Antibody (Alexa Fluor® 488 Conjugate).

4


p44/42 MAPK and MEK1/2

Phospho-MEK1/2 (Ser217/221)

Even

ts

Flow cytometric analysis of Jurkat cells,untreated (blue) or PMA treated (green),using Phospho-MEK1/2 (Ser217/221)Antibody compared to a nonspecificnegative control antibody (red).

Phospho-MEK1/2 (Ser217/221) Antibody #9121

Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mAb (AlexaFluor® 488 Conjugate) #4374

Even

ts

11

7

Phospho-p44/42 (Thr202/Try204)

Phospho-p44/42 MAPK (Thr202/Tyr204) (197G2) Rabbit mAb #4377

Western blot analysis of purified MAPKphospho-proteins or extracts fromNIH/3T3 cells treated with UV light andPDGF, using Phospho-p44/42 MAPK(Thr202/Tyr204) (197G2) Rabbit mAb.

Flow cytometric analysis of ungated human peripheral blood lymphocytes treated witheither the MEK inhibitor U0126 (left) or PMA (right), using Phospho-p44/42 MAPK(Thr202/Tyr204) (E10) Mouse mAb (Alexa Fluor®488 Conjugate).

Side Scatter

U0126-inhibited

Phos

pho-

p44/

42 M

APK

(Ale

xa F

luor

® 4

88) 103

102

101

100

0 1023

Side Scatter

PMA-stimulated

103

102

101

100

0 1023

Flow cytometric analysis of U0126-inhibited (blue) or PMA-stimulated(green) Jurkat cells, usingPhospho-p44/42 MAPK(Thr202/Tyr204) (197G2) RabbitmAb, compared to a nonspecificnegative control antibody (red).

MAP Kinases play a critical role in the regulation of cell growth, differ-entiation and stress response. MAP Kinases are activated by a wide vari-ety of extracellular signals including growth and neurotrophic factors,cytokines, hormones and neurotransmitters. Activation of ERK1/2 occursthrough phosphorylation of Thr202/Tyr204 by MEK1/2. ERK1/2 phospho-rylation is a common result of growth factor receptor activation or expo-sure to an oncogenic agent. When conducting flow cytometric analysisof cell signaling in normal or disease processes the MAP kinases areoften considered as possible key regulators.

A cell’s response to internal or external stress is intimately linked to pro-liferation and survival. Central to the stress-induced kinase cascades areSAPK/JNK, activated by phosphorylation at Thr183/Tyr185 and p38MAPK, activated by phosphorylation at Thr180/Tyr182. Activation ofthese signaling pathways results in transcriptional regulation of proteinsinvolved in diverse cellular processes including differentiation, apoptosis,cytokine production and cytoskeletal reorganization. Therefore, a flowcytometric analysis of the MAP kinases may be combined with an analy-sis of cell cycle, apoptosis and proliferation to determine the proximalevents leading to downstream changes in cell behavior.

nucleus

membraneRAS

Cytoplasm

Growth/NeurotrophicFactors

MEK1/2

Transcription

Ser259

Ser217/221

Thr202/Tyr204

Tyr1068

EGF

Ser383Thr58/Ser62

SRETranscription

GRBSOS

Ser380

Thr359Ser363

Ser338 Akt

c-Raf

PD98059

EGFR

MAPK/Erk1/2

U0126

p90RSK c-Myc Elk1

Even

ts

Phospho-SAPK/JNK (Thr183/Tyr185)(Alexa Fluor® 647)

Flow cytometric analysis of THP-1cells, untreated (blue) or anisomycin-treated (green), using Phospho-SAPK/JNK (Thr183/Tyr185) (G9)Rabbit mAb (Alexa Fluor® 647Conjugate) compared to a nonspecificcontrol antibody (red).

Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Rabbit mAb(Alexa Fluor® 647 Conjugate) #9257

Flow cytometric analysis of Jurkat cells,untreated (green) or anisomycin-treated(blue), using Phospho-SAPK/JNK(Thr183/Tyr185) (G9) Mouse mAb com-pared to a nonspecific negative controlantibody (red).

Phospho-SAPK/JNK (Thr183/Tyr185)(G9) Mouse mAb #9255

SAPK/JNK

nucleus

RASGRB

SOS

GF

Cytoplasm

Growth Factors

GF-RRac cdc42

SEK1/MKK4

SAPK/JNK

Thr69/71

ATF-2

CRE

Transcription Factors

Transcriptionc-Jun

AP-1

Ser63/73

Genotoxic Agents,Anisomycin

Thr261Ser80 Ser257

UVlight

Inflammatory Cytokines,TNF-α, IL-1

GCKs

ASK1MLKsMEKK1/4

Thr183/Tyr185

kDa

10080

6050

40

30

Phospho-p44 MAPK

Phospho-p42 MAPK

140200

Erk2 p38 JNK2 – + UV + PDGF

Recombinant NIH Protein 3T3

Phospho-SAPK/JNK (Thr183/Tyr185)

Even

ts


5



nucleus

SRE CRE/TRE CRE/TRE

RAS

GRB SOS

Growth Factors

InflammatoryCytokines

TNF-α IL-1

RTK Rac1

Cytoplasm

UV light

cdc42

TranscriptionTranscriptionFactors

Ser383

p38 MAPK

MKK6

Ser189 Ser207

Thr180/Tyr182

Thr69/71

Ser82 Thr197/Thr202

Ser360/376

MSK-1

Ser133ATF-2Elk-1 CREBMEF2

Thr581Thr312/319

TranslationMNK

MLK3

Thr277/Ser281

MKK3

MAPKAPK-2HSP27

Ser222 Thr334

Osmotic ShockGenotoxic Agents

Anisomycin

Flow cytometric analysis of untreated (left) and anisomycin-treated (right) human peripheralblood mononuclear cells, using Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb(Alexa Fluor® 647 Conjugate). Anisomycin treatment increases Phospho-p38 signal in asubpopulation of CD3-negative cells (box).

CD3

103

102

101

100

103102101100

CD3

Phos

pho-

p38

(Ale

xa F

luor

® 6

47)

103

102

101

100

103 102 101 100

Even

ts

58

Phospho-p38 MAPK (Alexa Fluor® 488)

Flow cytometric analysis of THP-1cells, untreated (blue) or anisomycin-treated (green), using Phospho-p38MAPK (Thr180/Tyr182) (28B10) MousemAb (Alexa Fluor® 488 Conjugate)compared to a nonspecific control anti-body (red).

Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb (AlexaFluor® 488 Conjugate) #4551

Phospho-p38 MAPK

Even

ts

Flow cytometric analysis of Jurkat cells, untreat-ed (green) or anisomycin treated (blue), usingPhospho-p38 MAP Kinase (Thr180/Tyr182)Antibody compared to a nonspecific negativecontrol antibody (red).

Phospho-p38 MAP Kinase (Thr180/Tyr182) Antibody #9211

Thr421

Ser424

Ser235Ser236 Ser240

Ser244

Thr229

Thr389

Raf PI3K

Erk1/2 PDK1 Akt

mTOR/FRAP

Growth Factors

Translation

rpS6

p70S6K

Even

ts

Phospho-p38 MAPK (Alexa Fluor® 647)

Flow cytometric analysis of THP-1 cells, untreat-ed (blue) or anisomycin-treated (green), usingPhospho-p38 MAPK (Thr180/Tyr182) (28B10)Mouse mAb (Alexa Fluor®647 Conjugate) com-pared to a nonspecific control antibody (red).

Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb(Alexa Fluor® 647 Conjugate) #4552

Phospho-S6 Ribosomal Protein (2F9) Rabbit mAb(Alexa Fluor® 488 Conjugate) #4854

Phospho-S6 Ribosomal Protein

Even

ts

Flow cytometric analysis of NIH/3T3 cells, U0126- andrapamycin-treated (blue) or serum-treated (green), usingPhospho-S6 Ribosomal Protein (Ser240/244) comparedto a nonspecific negative control antibody (red).

Phospho-S6 Ribosomal Protein (Ser240/244) Antibody #2215

p38 MAPK

S6 Ribosomal Protein

▲

Even

ts

Phospho-S6 Ribosomal ProteinAlexa Fluor® 488

Flow cytometric analysis of Jurkat cells, untreated (green),or LY294002, Wortmannin and U0126-treated (blue), usingPhospho-S6 Ribosomal Protein (Ser235/236) (2F9) RabbitmAb (Alexa Fluor® 488 Conjugate).

Even

ts

Phospho-Akt (Ser473) Alexa Fluor® 488

6


P P PP P PP PP P P

membrane

Akt/PKB

GSK3bTSC2 BAD

MDM2

FKHR

eNOS NO synthesis

Ser166/186

AFX

Receptor

Growth factors, insulin, etc.

p21CIP1

survivalgrowth

Ser241

Thr308

Ser473

Akt/PKB

Thr308

Ser473

Ser1177

Ser9 Ser136

Thr145/Ser146

Ser256 Thr24/32

glycogensynthesis

proliferationsurvival

proliferationsurvival

Thr1462

Ser939

Ser193

P13KPIP3

PIP3

Tyr376

Tyr373

PTEN

PDK1

SHIP

Phospho-Akt (Ser473) (193H12) Rabbit mAb (Alexa Fluor® 488 Conjugate) #2336

Western blot analysis of extracts fromNIH/3T3 cells, untreated, PDGF-treatedor PDGF-treated after treatment withwortmannin or rapamycin as indicated,using Phospho-Akt (Thr308) (244F9)Rabbit mAb (upper) or Akt Antibody#9272 (lower).

Cellular Survival: AktThe balance between apoptosis and cell survival is vital for main-taining tissue homeostasis. Aberrant control of apoptotic signalingcan result in tumor generation and resistance to chemopreventiveagents. There are multiple controls involved in this process.Therefore a multiplex analysis of disease cells is highly valuable.Such an analysis is possible using flow cytometry and activation-specific antibodies.

Akt, a serine/threonine protein kinase existing in mammals has atleast three known isoforms (Akt1, 2 and 3) and plays a critical rolein controlling this balance. As a result of signaling by insulin andvarious growth and survival factors, Akt is activated by phosphory-lation at Thr308 and Ser473. Akt then promotes cell survival byphosphorylating downstream targets, including Bad, forkhead tran-scription factors and caspase-9. Activated Akt and downstream sig-naling molecules can be simultaneously detected using flow cytom-etry.

Phospho-Akt (Thr308)

200140

100

80

60

50

40

30

60

50

40

30

200 140

100

80

kDa

Akt

Rapamycin (5 nM)

–– –– – –

–+ +

++

+

PDGF (50 ng/ml)Wortmannin (200 nM)

Even

ts

Phospho-Akt (Thr308)

Flow cytometric analysis of Jurkat cells, untreated(green) or LY294002 and wortmannin treated(blue), using Phospho-Akt (Thr308) (244F9)Rabbit mAb compared to a nonspecific negativecontrol antibody (red).

Phospho-Akt (Thr308) (244F9) Rabbit mAb #4056

Flow cytometric analysis of Jurkat cells,untreated (green) or LY294002 and wort-mannin treated (blue), using Phospho-Akt(Ser473) (193H12) Rabbit mAb (AlexaFluor® 488 conjugate).

Survival/Apoptosis

▲

Phospho-Akt (Ser473) (193H12) Rabbit mAb (Alexa Fluor® 647 Conjugate) #2337

Even

tsPhospho-Akt (Ser473) Alexa Fluor® 647

Flow cytometric analysis of Jurkat cells,untreated (green) or LY294002 and wort-mannin treated (blue), using Phospho-Akt(Ser473) (193H12) Rabbit mAb (AlexaFluor® 647 conjugate).

Phospho-Akt (Ser473) (193H12) Rabbit mAb #4058

Even

ts

Phospho-Akt (Ser473)

Flow cytometric analysis of Jurkat cells,untreated (green) or LY294002 andWortmannin treated (blue), usingPhospho-Akt (Ser473) (193H12) RabbitmAb compared to a nonspecific negativecontrol antibody (red).

Akt Antibody #9272

Akt Antibody

Even

ts

Flow cytometric analysis of Jurkat cells,untreated (blue) or LY294002-treated(green), using Akt Antibody compared to anonspecific negative control antibody (red).

7


Survival/Apoptosis

membrane

Cytoplasm

FasL, TNF-α

Death stimuli

[Ca ]++

ER Stress

Apoptosis

α-Fodrin

Smac/Diablo

Caspase-8/10

Caspase 3

Ser194

Lamin A DFF

Caspase-7Caspase-6

XIAP,SurvivinCaspase-12 Caspase-9

PARP

Cyto c

FADD

Cleaved Caspase-3

Even

ts

Flow cytometric analysis of Jurkat cells,untreated (blue) or etoposide-treated(green), using Cleaved Caspase-3(Asp175) Antibody compared to a non-specific negative control antibody (red).

Cleaved Caspase-3 (Asp175) Antibody #9661

Even

ts

Cleaved Caspase-3 Alexa Fluor®

488

Flow cytometric analysis of Jurkat cells,untreated (blue) or etoposide-treated(green), using Cleaved Caspase-3(Asp175) Antibody (Alexa Fluor®488Conjugate).

Cleaved Caspase-3 (Asp175) (Alexa Fluor® 488 Conjugate) #9669

Cleaved Caspase-9

Even

ts

Flow cytometric analysis of Jurkatcells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-9 (Asp330) Antibody (Human Specific)compared to a nonspecific negativecontrol antibody (red).

Cleaved Caspase-9 (Asp330) Antibody (Human Specific) #9501

Cleaved Caspase-9 (Asp330) (37 kDa)

kDa

Procaspase-9 (47 kDa)

46.5

28

57

46.5

0 1.5 3 5 0 3 5 Treatment (hrs)

HeLa Jurkat

Staurosporine Etoposide

Western blot analysis of HeLa cells, untreated or staurosporine-treated (1 µM),and Jurkat cells, untreated or etoposide-treated, using Cleaved Caspase-9(Asp330) Antibody (Human Specific) (upper) or full length Caspase-9 Antibody#9502 (lower).

Cleaved Caspase-7

Even

ts

Cleaved Caspase-8

Even

ts

Flow cytometric analysis of Jurkat cells,untreated (blue) or etoposide-treated(green), using Cleaved Caspase-7(Asp198) Antibody compared to a non-specific negative control antibody (red).

Flow cytometric analysis of Jurkatcells, untreated (blue) or etoposide-treated (green), using CleavedCaspase-8 (Asp374) (18C8) RabbitmAb compared to a nonspecific nega-tive control antibody (red)..

Cleaved Caspase-7 (Asp198) Antibody #9491

Apoptosis: Cleaved CaspasesCysteine aspartic acid proteases (caspases) are key executioners ofapoptosis. Upon apoptotic stimulation, initiator caspases, includingcaspase-9, are activated by proteolysis. They then process and acti-vate effector caspases, including caspase-3 and –7, which amplifythe apoptotic cascade. Effector caspases are responsible for thecleavage of a wide range of substrates, including protein kinases,other signaling proteins and many structural proteins.Immunodetection of cleaved caspases is a straightforward andwidely accepted means of identifying cells undergoing apoptosisand flow cytometry is a powerful technique for accomplishing this.This assay has the advantage over other assays such as Annexin Vstaining due to it’s specificity for apoptosis.

Cleaved Caspase-8 (Asp374) (18C8) Rabbit mAb #9496

8


Jak/Stat Signaling

Jak/Stat Pathway

Tyk2 JAKS

STATS STATS

STATS

STATS

RafPI3K

Akt

ErkmTOR

TranscriptionFactors

nucleus

cytoplasm

membrane

ISRE / GAS

Transcription

Cytokines


Even

ts

Flow cytometric analysis of HeLa cellsuntreated (blue) or IFN-α treated (green),using Phospho-Stat1 (Tyr701) Antibodycompared to a nonspecific negative controlantibody (red).

Phospho-Stat1 (Tyr701) Antibody #9171

Stat1

Even

ts

Flow cytometric analysis of HeLa cellsuntreated (blue) or IFN-α treated (green),using Stat1 Antibody compared to a nonspe-cific negative control antibody (red).

Stat1 Antibody #9172

Flow cytometric analysis of HeLacells, untreated (blue) or IFN-α−treated (green), using Phospho-Stat3 (Tyr705) (D3A7) Rabbit mAb.

Phospho-Stat3 (Tyr705) (D3A7) Rabbit mAb #9145


Even

ts

Even

ts


Flow cytometric analysis of HeLa cellsuntreated (blue) or IFN-α treated(green), using Phospho-Stat3 (Tyr705)Antibody, compared to a nonspecificnegative control antibody (red).



Even

ts

Flow cytometric analysis of Ramoscells untreated (blue) or IL-4 treated(green), using Phospho-Stat6 (Tyr641)Antibody, compared to a nonspecificnegative control antibody (red).


Jaks and Stats are critical components of many cytokine receptor sys-tems, regulating growth, survival, differentiation and pathogen resist-ance. When phosphorylated and activated by Jaks, Stats are trans-ported to the nucleus where they regulate cytokine-induced geneexpression. Therefore, the Jak/Stat pathways are of great importanceto cellular signaling in hematopoietic cells whose proliferation is reg-ulated by cytokines.

Stat1, inappropriately activated in many tumors, appears to beessential for responsiveness to IFN-α and IFN-γ. Stat1 is activatedby phosphorylation at Tyr701.

Stat3 can function as an oncogene and is constitutively active in manycancers. It is activated by many cytokines and growth factor receptorsthrough phosphorylation at Tyr705.

Stat5 is activated in response to a wide variety of ligands includingIL-2, GM-CSF, growth hormone and prolactin. Transformation withBcr-Abl causes constitutive Tyr694 phosphorylation and activationof Stat5, a condition also seen in some leukemic cell types. Stat5also appears to be an important downstream substrate for the FLT3receptor.

Stat6 is activated via phosphorylation at Tyr641 and is required forresponsiveness to IL-4 and IL-13. In addition to its role in mediatingIL-4 responses, Stat6 is activated in B cell lines by IFN-α.

Clearly, any flow cytometric analysis of cell signaling in hematopoi-etic cells may also require a consideration of the Jak/Stat pathways.

9


Cyclin D cdk4

Thr373

Ser780

Ser608

Ser795

Ser807/811Ser249/252

Restriction Point

DNA Synthesis

+

S G1

ExtracellularSignals

Cyclin A cdk2

Cyclin E cdk2 Rb Rb E2F

Phospho-Histone H3 (Ser10)

Even

ts

Cell Cycle / DNA Damage

Phos

pho-

Rb

(Ser

807/

811)

DNA (PI)

103

102

101

100

0 1023

Flow cytometric analysis of Phospho-Rb(Ser807/811) staining versus DNA content(propidium iodide) in untreated Jurkat cells,using Phospho-Rb (Ser807/811) Antibody.Box indicates phospho-Rb positive cells.

Phospho-Rb (Ser807/811) Antibody #9308

Cyclin D1 (DCS6)

Even

ts

Flow cytometric analysis of Cyclin D1 (DCS6)Mouse mAb staining of A431 cells (blue) comparedto a nonspecific negative control antibody (red).

Cyclin D1 (DCS6) Mouse mAb #2926

Phos

pho-

His

tone

H3

(Ser

10) A

lexa

Flu

or®

488

Cyclin B1 (V152) Alexa Fluor® 647

103

102

101

100

100 101 102 103

Two-color flow cytometric analysis ofasynchronous Jurkat cells, using CyclinB1 (V152) Alexa Fluor® 647 Conjugateand Phospho-Histone H3 (Ser10) AlexaFluor® 488 Conjugate (#9708). Cellsrepresented in green are positive forCyclin B1 and Phospho-Histone H3,while cells represented in blue are posi-tive for Phospho-Histone H3 and nega-tive for Cyclin B1. Both cell populations(green and blue) correspond to cellsundergoing mitosis.

Cyclin B1 (V152) Mouse mAb (Alexa Fluor® 647 Conjugate) #4118

Cyclin D1

kDa HeLa 3T3

40

140100

80

50

30

60

Western blot analysis of extractsfrom HeLa and NIH/3T3 cells, usingCyclin D1 (DCS) Mouse mAb.

Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706

Phospho-Histone H3

Even

ts

Flow cytometric analysis of Ramos cellsuntreated (blue) or serum/calyculin treated(green), using Phospho-Histone H3 (Ser10)Antibody compared to a nonspecific negativecontrol antibody (red).

Phospho-Histone H3 (Ser10) Antibody #9701

Phos

pho-

His

tone

H3

(Ser

10)

DNA (PI)

103

102

101

100 Flow cytometric analysis of untreatedJurkat cells, using Phospho-HistoneH3 (Ser10) Antibody versus propidi-um iodide (DNA content). The boxindicates Phospho-Histone H3 posi-tive cells.

▲

nuclear membrane

Aurora B

PP1DNA

Mitosis(Chromatin condensation)

Interphase(Chromatin decondensation)

Growth Factors/Cytokine

Phos

Phos

Phos Phospho-group Histone tail DNA

Nucleosome core Acetyl group

ACAC

AC

AC

AC

▲

Phos

pho-

His

tone

H3

(Ser

10)

103

102

101

100

0 1023 DNA (PI)

A. Flow cytometry analysis of Phospho-Histone H3 (Ser10) (6G3) Mouse mAb stainingversus DNA content (propidium iodide) in paclitaxel treated THP-1 cells. Red popula-tion indicates Phospho-Histone H3 positive cells.

B. Flow cytometric analysis of Phospho-Histone H3 (Ser10) (6G3) Mouse mAb stain-ing of untreated (blue) or serum/calyculin treated (green) Ramos cells compared to anonspecific negative control antibody (red).

A B

Cell Cycle Progression through the cell cycle is a tightly controlled process,and disregulation of this process is associated with tumor genera-tion and survival. Cell cycle regulatory proteins have long beentargets of anti-cancer therapies. The use of DNA staining and cellcycle analysis is a central part of flow cytometry, and the additionof intra-cellular staining and activation-specific antibodies nowallows for the flow cytometric analysis of the proteins regulatingthe cell cycle.

The phosphorylation state of the retinoblastoma protein (Rb) iswidely relied upon as a marker of G1/S phase transition. Cyclin D1 issynthesized when cells enter the cell cycle, and its presence isindicative of proliferating cells.

Phosphorylation of Histone H3 at Ser10 is linked to the initiation ofchromosome condensation and is considered to be a marker forcells in mitosis. Flow analysis can be used to examine these andother important targets and has been used to identify many addi-tional phosphorylation events associated with cell cycle progression.

10


Even

ts

Phospho-Histone H2A.X (Ser139)

Flow cytometric analysis of HeLacells, untreated (blue) and UV-treat-ed (green), using Phospho-HistoneH2A.X (Ser139) Antibody comparedto a nonspecific negative controlantibody (red).

Phospho-Histone H2A.X (Ser139) Antibody #2577

DNA Damage

Even

ts

Phospho-H2A.X (Ser139) Alexa Fluor® 488

Flow cytometric analysis of Jurkatcells, untreated (green) or etoposide-treated (blue), using Phospho-HistoneH2A.X (Ser139) (20E3) Rabbit mAb(Alexa Fluor® 488 Conjugate).

Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 488 Conjugate) #9719 Phospho-Chk1 (Ser345) Antibody #2341

Cell Cycle / DNA Damage

Phos

pho-

Chk2

(Thr

68)

DNA (PI)

103

102

101

100

0 1023

Flow cytometric analysis of untreatedJurkat cells, using Phospho-Chk2(Thr68) Antibody versus propidiumiodide (DNA content). The boxedpopulation indicates phospho-Chk2(Thr68)-positive cells.

Phos

pho-

Chk1

(Ser

345)

DNA (PI)

103

102

101

100

0 1023

Flow cytometric analysis of untreatedJurkat cells, using Phospho-Chk1(Ser345) Antibody versus propidiumiodide (DNA content). The box indi-cates phospho-Chk1 (Ser345)-posi-tive cells.

In the past, analyses of cellular signaling in whole blood has been

complicated by the fact that traditional flow cytometric

fixation/permeabilization protocols for immunophenotyping with

surface markers did not work well with signaling antibodies direct-

ed against intracellular targets. Most whole blood protocols

include a lysis step prior to fixation, but some signaling events are

very transient and might be missed in the time it takes to lyse ery-

throcytes. In addition, the lysis step may induce artifactual signal-

ing events. Many commercial "fix & perm" kits do not contain

enough fixative to stabilize phospho epitopes and deactivate phos-

phatases, and they may not adequately permeabilize the cells.

Permeabilization with 90-100% methanol works very well with

intracellular signaling antibodies (Krutzik and Nolan, 2003), but

methanol permeabilization may diminish or abolish signal from sur-

face markers and alters the scatter characteristics of cells. Chow et

al. (2005) recently published a new protocol (summarized below)

that utilizes a combination of formaldehyde, Triton X-100, and 50%

methanol. This new protocol allows for the concurrent use of intra-

cellular phospho-specific antibodies and surface markers, and also

preserves cell scatter characteristics. This should be useful in the

analysis of complex cellular signaling in whole blood, marrow, aspi-

rates, effusions or other fluids containing blood cells.

Krutzik P.O. and Nolan G.P. (2003) Intracellular phospho-protein

staining techniques for flow cytometry: monitoring single cell sig-

naling events. Cytometry A. 55, 61-70.

Chow, S. et al (2005) Whole blood fixation and permeabilization

protocol with red blood cell lysis for flow cytometry of intracellular

phosphorylated epitopes in leukocyte subpopulations. Cytometry A.

67, 4-17.

Phospho-Chk2 (Thr68) Antibody #2661

Flow Protocols

DNA damage caused by ionizing radiation, UV-light or radiomimetic agents results in rapid phosphorylation of the histone H2A familymember H2A.X at Ser139 by ATM. Within minutes following DNA damage, Ser139-phosphorylated H2A.X localizes to sites of DNA damageat subnuclear foci.

Chk1, one of the downstream protein kinases of ATM/ATR, plays an important role in DNA damage checkpoint control, embryonic develop-ment and tumor suppression. The activation of Chk1 in response to blocking DNA replication and certain forms of genotoxic stress involvesphosphorylation of Ser317 and Ser345. Chk1 is also phosphorylated at Ser280 and Ser296 following DNA damage. Activated Chk1 caninactivate cdc25C, blocking the activation of cdc2 and transition into mitosis.

A. Solutions and Reagents 1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl,

1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O).Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at roomtemperature.

2. Formaldehyde (methanol free) 3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in

100 mL 1X PBS. Store at 4°C

B. Preparation of Whole Blood (fixation, lysis and permeabilization) forImmunostaining

1. Aliquot 100 ul fresh whole blood per assay tube. 2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term

treatments with ligands, inhibitors, drugs, etc. 3. Add 65 ul of 10% formaldehyde to each tube. 4. Vortex briefly and let stand for 15 minutes at room temperature. 5. Add 1 ml of 0.1135% Triton X-100 in PBS (0.1% Triton final). 6. Vortex and let stand for 30 minutes at room temperature. 7. Add 1 ml Incubation Buffer. 8. Pellet cells by centrifugation and aspirate supernatant. 9. Resuspend cells in cold 50% methanol in PBS (store methanol

solution at -20°C until just before use). 10. Incubate at least 10 minutes on ice. 11. Proceed with staining or store cells at –20°C in 50% methanol.

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies1. Add 1 ml Incubation Buffer to each tube and rinse by

centrifugation. Repeat. 2. Add primary antibodies diluted as recommended on data sheet in

Incubation Buffer. 3. Incubate for 30-60 minutes at room temperature. 4. Rinse as before in Incubation Buffer by centrifugation.

5. Resuspend cells in fluorochrome-conjugated secondary antibody*diluted in Incubation Buffer according to the manufacturer’srecommendations.

6. Incubate for 30 minutes at room temperature. 7. Rinse as before in Incubation Buffer by centrifugation. 8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

*Recommended Secondary Antibodies from Invitrogen: A-11070 Alexa Fluor®488 F(ab)2 fragment of goat anti-rabbit IgG (H+L) (1:1000 dilution) andA-11017 Alexa Fluor® 488 F(ab)2 fragment of goat anti-mouse IgG (H+L)1:1000 dilution).

Chow, S. et al (2005) Whole blood fixation and permeabilization protocolwith red blood cell lysis for flow cytometry of intracellular phosphorylatedepitopes in leukocyte subpopulations. Cytometry A. 67, 4-17.

11


Flow cytometric analysis of untreated or PMA-stimulated normal human peripheralblood, using phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mAb. Peripheralblood was prepared according to the protocol of Chow et al. (2005). Data kindly provid-ed by Sue Chow and David Hedley, Princess Margaret Hospital, Toronto.

Alternate Protocol for Signaling Analysis in Peripheral Blood

Untreated PMA-treated

Flow Cytometry Protocol Using Activation-state Antibodies Solutions and Reagents1X Phosphate Buffered Saline (PBS):

Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.

Formaldehyde (methanol free)Incubation Buffer:

Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.

Fixation1. Collect cells by centrifugation and aspirate supernatant. 2. Resuspend cells briefly in 0.5-1 ml PBS, then add formaldehyde so

that the final concentration is % formaldehyde.3. Fix for 10 minutes at 37°C.4. Chill tubes on ice for 1 minute.

Permeabilization1. Permeabilize cells by adding ice-cold 100% methanol slowly to

pre-chilled cells while gently vortexing so that the final concentra-tion is 90% methanol. Alternatively, to remove fix prior to perme-abilization, pellet cells by centrifugation and resuspend in 90% methanol.

2. Incubate 30 minutes on ice or at 4°C. 3. Proceed with staining or store cells at –20°C in 90% methanol.

StainingNote: Allow for isotype matched controls for monoclonal antibodies orrabbit IgG for polyclonal antibodies.

1. Count cells using hemacytometer or alternative method.2. Aliquot 0.5-1 x 106 cells into each assay tube (by volume).3. Add 2-3 ml Incubation Buffer to each tube and rinse by centrifu-

gation. Repeat.4. Resuspend cells in 100 µl Incubation Buffer per assay tube.5. Let cells block in Incubation Buffer for 10 minutes at room

temperature. 6. Add the primary antibody at the appropriate dilution to the assay tubes

(see datasheet of each antibody for the appropriate dilution).7. Incubate for 30-60 minutes at room temperature. 8. Rinse as before in Incubation Buffer by centrifugation.9. Resuspend cells in fluorochrome-conjugated secondary antibody,*

diluted in Incubation Buffer per manufacturer’s recommendations.10. Incubate for 30 minutes at room temperature. 11. Rinse as before in Incubation Buffer by centrifugation.12. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

*Recommended Secondary Antibodies from InvitrogenA-11070 Alexa Fluor® 488 F(ab')2 fragment of goat anti-rabbit IgG (H+L) (1:1000 dilution)

A-11017 Alexa Fluor® 488 F(ab')2 fragment of goat anti-mouse IgG (H+L)(1:1000 dilution)

Flow Protocols

Publications using Phospho antibodies for Flow CytometryChow, S. et al. (2001) Measurement of MAP kinase activation byflow cytometry using phospho-specific antibodies to MEK and ERK:potential for pharmacodynamic monitoring of signal transductioninhibitors. Cytometry 46, 72–8.

Perez, O.D. and Nolan, G.P. (2002) Simultaneous measurement ofmultiple active kinase states using polychromatic flow cytometry.Nat. Biotechnol. 20, 155–62.

Rovida, E. et al. (2002) Opposite effects of different doses of MCSFon ERK phosphorylation and cell proliferation in macrophages.Oncogene 21, 3670–6.

Weiss, L. et al. (2002) Regulation of c-Jun NH(2)-terminal kinase(Jnk) gene expression during T cell activation. J. Exp. Med. 191,139–46.

Tazzari, P.L. et al. (2002) Flow cytometric detection of total and ser-ine 473 phosphorylated Akt. J. Cell. Biochem. 86, 704–15.

Cavallo, F. et al. (2002) Interleukin 12-activated lymphocytes influ-ence tumor genetic programs. Cancer Res. 61, 3518–23.

Cortez, D. et al. (2002) ATR and ATRIP: partners in checkpoint sig-naling. Science 294, 1713–6.

Ilangumaran, S. et al. (2003) Flow cytometric analysis of cytokinereceptor signal transduction. J. Immunol. Methods 278, 221–34.

Banath, J.P. and Olive, P.L. (2003) Expression of phosphorylated his-tone H2AX as a surrogate of cell killing by drugs that create DNAdouble-strand breaks. Cancer Res. 63, 4347–50.

Perez, O.D. et al. (2003) Leukocyte functional antigen 1 lowers Tcell activation thresholds and signaling through cytohesin-1 andJun-activating binding protein 1. Nat. Immunol. 4, 1083–92.

Jacobberger, J.W. et al. (2003) Immunoreactivity of Stat5 phospho-rylated on tyrosine as a cell-based measure of Bcr/Abl kinase activ-ity. Cytometry 54A, 75–88.

Krutzik, P.O. et al. (2004) Analysis of protein phosphorylation andcellular signaling events by flow cytometry: techniques and clinicalapplications. Clin. Immunol. 110, 206–21.

Grammer, A.C. et al. (2004) Flow cytometric assessment of the sig-naling status of human B lymphocytes from normal and autoim-mune individuals. Arthritis Res. Ther. 6, 28–38.

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FLOW CYTOMETRY - korambiotech.comkorambiotech.com/upload/bbs/2/Cytometry.pdfTo this end, flow cytometry has been widely applied to the semi- quantitative analysis of mixed populations