i h h h l i l d fl i i i llHigh Throughput Multiplexed Inflammation Assay Using Primary CellsHigh-Throughput Multiplexed Inflammation Assay Using Primary CellsHigh-Throughput Multiplexed Inflammation Assay Using Primary CellsHigh Throughput Multiplexed Inflammation Assay Using Primary Cellsg g p p y g yOk Si k Y W Ch J H l C h Ol H R T E F C ll* Oksana Sirenko Yen-Wen Chen Jayne Hesley Cathy Olsen H Roger Tang Evan F Cromwell* Oksana Sirenko, Yen-Wen Chen, Jayne Hesley, Cathy Olsen, H. Roger Tang, Evan F. Cromwell* , , y y, y , g g,

Molecular Devices Inc 1311 Orleans Drive Sunnyvale CA 94089Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089

Inhibition of Adhesion Molecules by Anti-Inflammatory High-Throughput Assay for Anti-Inflammation Efficacy of CompoundsI t d ti R lt d di i

Inhibition of Adhesion Molecules by Anti-Inflammatory High-Throughput Assay for Anti-Inflammation Efficacy of CompoundsIntroduction Results and discussion CompoundsIntroduction Results and discussion Compounds

An important event in the inflammation response is the expression of Expression of Inflammation Markers Measured by IsoCyte A ti t f i fl tiA ti t f i fl tiDose-dependent inhibition of VCAM expression by SB202190: An important event in the inflammation response is the expression of Expression of Inflammation Markers Measured by IsoCyte

Inhibitors of Activators of inflammationInhibitors of Activators of inflammationp p y

cell-surface antigens that facilitate binding of immune cells to blood Inhibitors of Inhibitors of cell surface antigens that facilitate binding of immune cells to blood Inflammation:Inflammation:vessels The ability to monitor up-regulation of these molecules such

Inflammation:Inflammation:vessels. The ability to monitor up regulation of these molecules, such

VCAM E l i d HLADR d h li l ll id as VCAM, E-selectin, and HLADR on endothelial cells provides an LPSLPSAlexa - CD31 APC - HLA-DRas VCAM, E selectin, and HLADR on endothelial cells provides an i t t h i l i l d t f ll b d d l f TNF IL-1 LPSTNF IL-1 LPSAlexa CD31 APC HLA DR

important physiological read-out for cell-based models of TNF IL 1TLR4

IFNTNF IL 1

TLR4IFN1.2E+10 3.0E+08important physiological read out for cell based models of

i fl ti TLR4

antibodies TLR4

antibodies2 5E 08inflammation. CD14antibodies CD14antibodies

1.0E+10 2.5E+08

0 uM 1 uM 3 uM 10 uM 30 uM8 0E 09

2 0E+08

We present results from a multiplexed primary human cell based TRADD MyoD88TOLLIPTRADD MyoD88TOLLIP8.0E+09 2.0E+08

I

We present results from a multiplexed, primary human cell-based TRADD MyoD88kinase

TRADD MyoD88kinase VCAM-red; CD31-green6 0E+09FI

1.5E+08FIp p , p yassay that uses fluorescence read outs of inflammation markers to IRAKRIP JAKkinase IRAKRIP JAKkinase ; g6.0E+09

assay that uses fluorescence read-outs of inflammation markers to MEKK VCAMinhibitors MEKK VCAMinhibitors VCAM intensity of fluorescence (heat-map):4.0E+09 1.0E+08

evaluate the effect of different mediators on inflammatory response NIKMEKK VCAM

E selinhibitors

NIKMEKK VCAM

E selinhibitors VCAM, intensity of fluorescence (heat-map):

5 0E+07evaluate the effect of different mediators on inflammatory response. NIKMEKK3/6 E-selNIKMEKK3/6 E-sel Kinase inhibitors2.0E+09 5.0E+07

Expression of the inflammation markers on primary endothelial cells HLADRHLADR0 0E 00 0 0E+00Expression of the inflammation markers on primary endothelial cells IKK JNK HLADRStat1• Stimulation of cells with IKK JNK HLADRStat1• Stimulation of cells with +TNFNoTNF0.0E+00 0.0E+00

stimulated non-stim isotype(HUVEC) stimulated with inflammation cytokines (TNF or IFN) was

IKK JNKStimulation of cells with cytokines up-regulates

IKK JNKStimulation of cells with cytokines up-regulates

stimulated non-stim isotype stimulated non-stim isotype(HUVEC) stimulated with inflammation cytokines (TNF or IFN) was

ifi d b f l fl i i f IkBcytokines up regulates VCAM E-selectin HLADR IkBcytokines up regulates VCAM E-selectin HLADRquantified by measurement of total fluorescence intensity after IkBNFkBVCAM, E-selectin, HLADR

• Treatment with kinaseIkBNFkBVCAM, E-selectin, HLADR

• Treatment with kinasequantified by measurement of total fluorescence intensity after t i i ith di tl j t d tib di

• Treatment with kinaseinhibitors will decrease

• Treatment with kinaseinhibitors will decrease DMSO

PE - VCAM APC - E-selectinstaining with directly conjugated antibodies. inhibitors will decrease expression of inflammationinhibitors will decrease expression of inflammation

DMSOC

2 0E 10

APC - E-selectinstaining with directly conjugated antibodies. NFkB

expression of inflammation markers NFkBexpression of inflammation markers1 8 10

2.0E+106 0E+09

Comparison is made between IsoCyte scanning cytometer and NFkB AP1markers NFkB AP1markers

Compound 11.8E+10 6.0E+09

Comparison is made between IsoCyte scanning cytometer and • Anti-inflammatory efficacy • Anti-inflammatory efficacy Compound 11.6E+10 5.0E+09 p y g y

SpectraMax® M5 microplate reader for fold increase Z prime and of compounds could be of compounds could be 1.4E+10 4 0E+09

SpectraMax® M5 microplate reader for fold increase, Z-prime, and 38

tested in cell-based assay38

tested in cell-based assay Compound 21.2E+10FI 4.0E+09

p p , p ,

throughput The multiplexed read outs test the efficacy of anti p38p38

p38p38

Compound 21.0E+10

F

3.0E+09FIthroughput. The multiplexed read-outs test the efficacy of anti- kinasep38

kinasep38

8.0E+09

inflammatory compounds versus toxicity and also provide significant PKCkinase PKCkinaseCompound 36.0E+09 2.0E+09inflammatory compounds versus toxicity and also provide significant inhibitorsinhibitorsCompound 3

4.0E+09 1.0E+09

insight into the mechanism of action by selective inhibition of markers 2.0E+091.0E 09

insight into the mechanism of action by selective inhibition of markers Compound 40 0E+002.0E 09

0.0E+00

triggered by different signaling pathways C i f A ti I fl t P t f C d0.0E+00

stimulated non stim isotype stimulated non-stim isotypetriggered by different signaling pathways. Comparison of Anti-Inflammatory Potency of Compoundsstimulated non-stim isotype p y y pPE-VCAM APC HLADR

P i H I hibiti f VCAMVCAM-PE HLADR APCI hibiti f VCAMVCAM-PE HLADR APCPE-VCAM APC-HLADR

Primary Human Inhibition of HLA-DRInhibition of VCAMVCAM PE HLADR-APCInhibition of HLA-DRInhibition of VCAMVCAM PE HLADR-APC384 well format96 well formatIsoCyte 384 well format96 well formatIsoCyte 384 well format96 well formatSpectraMax® M5 384 well format96 well formatSpectraMax® M5

1.8E+10 4.5E+09yEndothelial Cells

2.5e8

C2.5e8

C384 well format96 well formatIsoCyte 384 well format96 well formatIsoCyte 384 well format96 well formatSpectraMax® M5 384 well format96 well formatSpectraMax® M51.6E+10 4.0E+09Endothelial Cells

7e9 PDTC7e9 PDTC S/C Z’S/C Z’ S/C Z’S/C Z’ S/C Z’S/C Z’ S/C Z’S/C Z’1.4E+10 3.5E+09 7e9

IC50 1860 uM7e9

IC50 1860 uM 580 0 768 0 75VCAM 580 0 768 0 75VCAM 5 0 7520 0 73VCAM 5 0 7520 0 73VCAM1.2E+10 3.0E+09

2e86e9IC50 1860 uMPDTC 2e86e9IC50 1860 uMPDTCPDTC 580 0.768 0.75VCAM 580 0.768 0.75VCAM 5 0.7520 0.73VCAM 5 0.7520 0.73VCAM1.0E+10FI iso

t2.5E+09

FI iso 6e9 PDTC

C6e9 PDTC

CPDTCC

12 0.823 0.72HLA-DR 12 0.823 0.72HLA-DR 4 0.7012 0.93CD31 (PECAM) 4 0.7012 0.93CD31 (PECAM)8.0E+09 st 2.0E+09

F st

5e9 IC50 183uM5e9 IC50 183uMIC50 183uM 156 0 766 0 81E selectin 156 0 766 0 81E selectin4 0.7012 0.93CD31 (PECAM) 4 0.7012 0.93CD31 (PECAM)

6.0E+09 1.5E+09

1 5e85e9

1 5e85e9 156 0.766 0.81E-selectin 156 0.766 0.81E-selectin4.0E+09 1.0E+09

TNF VCAM1.5e8

4 9

1.5e8

4 9 126 0.7374 0.93CD31 (PECAM) 126 0.7374 0.93CD31 (PECAM)2.0E+09 5.0E+08 S/C- signal to control, stimulated vs non-stimulated 4e94e9 126 0.7374 0.93CD31 (PECAM) 126 0.7374 0.93CD31 (PECAM)

0.0E+00PE 48h PE i 3AB 48h 3AB i

0.0E+00/ g ,

cells; for CD31 – signal to isotype controlIL 1 E selectin 3 93 9

PE 48h PE no stim 3AB 48h 3AB no stim APC 48h APC no stim 3AB 48h 3AB no stim cells; for CD31 signal to isotype controlor IL-1 E-selectin 1e83e9 1e83e9

Figure 1 HUVECs e e stim lated ith c tokines fo 24h Fl o escent AG126G AG126GGIFN HLA-DR Figure 1. HUVECs were stimulated with cytokines for 24h. Fluorescent 2e9 AG126AG1262e9 AG126AG126AG126I C S M ® MIFN intensities were measured by IsoCyte after staining cells with directly 5e7

AG1265e7

AG126AG126IsoCyte SpectraMax® M5

M lti lintensities were measured by IsoCyte after staining cells with directly

j t d tib di i t i t t l A i b t 1e9 IC50 32uMIC50 4.1uM1e9 IC50 32uMIC50 4.1uMIC50 4.1uM

y pVCAM inhibition VCAM inhibition

I fl tMultiple conjugated antibodies or appropriate isotype controls. A comparison between IC50 4.1uMIC50 4.1uMIC50 4.1uM VCAM inhibition

45

Inflammatory Readoutsj g pp p yp p

single AB (antibodies) and multiplexed AB (3AB) is shown in the bottom 00 00

PathwaysReadouts single AB (antibodies) and multiplexed AB (3AB) is shown in the bottom

h h ll l l d l d f d ff0.1 1 10 100 1000 10000

00.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000

00.1 1 10 100 1000 10000 2e9 40Pathways

charts. The assay allows multiplexing and simultaneous read-outs for different Concentration uMConcentration, uM Concentration uMConcentration, uMc a ts e assay a o s u t p e g a d s u ta eous ead outs o d e e tmarkers and signaling pathways

Concentration, uMConcentration, uM35markers and signaling pathways.

Figure 3 Tyrosine kinase inhibitor AG126 and anti oxidant PDTC both decrease 1.5e9

35

Figure 3. Tyrosine kinase inhibitor AG126 and anti-oxidant PDTC both decrease 30

h dup-regulation of VCAM and HLA-DR. IC50s for different compounds could be

30

Methodup regulation of VCAM and HLA DR. IC50s for different compounds could be dete mined b the assa 1e9 25Method

i f fl i k d b ldetermined by the assay. 1e9 25

Expression of Inflammation Markers Measured by Plate 20Expression of Inflammation Markers Measured by Plate R d S t M ® M5 5e8

20

SB202190 22 3 uMSB202190 18.0 uM

Fluorescence Detection of Inflammation Markers Reader SpectraMax® M5 5e8

15

SB202190 22.3 uMAG126 47 1 MAG126 40.1 uMFluorescence Detection of Inflammation Markers Reader SpectraMax M5

Multiplexing of Inflammation Assay15 AG126 47.1 uMAG126 40.1 uM

Multiplexing of Inflammation Assay0

Th I C t DL i t t l tf d f thi d t ti p g y

0.01 0.1 1 10 100 1000 100000

0.01 0.1 1 10 100 1000 1000010

The IsoCyte-DL scanning cytometer platform used for this demonstration was VCAM PE & CD31 AlexaVCAM PE & CD31 Alexa Concentration, uM Concentration, uMCD31VCAM

y g y pconfigured with a 20mW 488nm and 40mW 640nm lasers The 488nm laser was used M ultip le xing VCAM and CD31Multiplexing VCAM and CD31

VCAM-PE & CD31-Alexa M ultip le xing VCAM and CD31Multiplexing VCAM and CD31VCAM-PE & CD31-Alexa CD31 VCAMconfigured with a 20mW 488nm and 40mW 640nm lasers. The 488nm laser was used

f l l ( ) l b l d ll d b d (b ) 2e10

Multiplexing VCAM and CD31

2e10

Multiplexing VCAM and CD31

Figure 5 The assay is suitable for either 96 well or 384 well format Experimental windows 2035for Alexa Fluor® 488 (AF488) labeled cell detection using a 510-540nm band pass (bp) 2e102e10 2e102e10 Figure 5. The assay is suitable for either 96-well or 384-well format. Experimental windows 2035o e a uo ® 88 ( 88) abe ed ce detect o us g a 5 0 5 0 ba d pass (bp)filter (Ch1); and Phycoerythrin labeled detection of the markers using a 560 610nm bp VCAMVCAM were smaller for 384 format in comparison to 96 format. This could be due to instrumental 15

30filter (Ch1); and Phycoerythrin-labeled detection of the markers using a 560-610nm bp VCAM VCAMVCAM VCAM were smaller for 384 format in comparison to 96 format. This could be due to instrumental

diff h i th bi l i l l l f i f th k1525

filter (Ch3). APC detection was done at 640 excitation and 660-720nm emission filter. 1.5e101.5e10VCAM

1.5e101.5e10VCAM differences or changes in the biological levels of expression of the markers20filter (Ch3). APC detection was done at 640 excitation and 660 720nm emission filter.

The image acquisition was done at 5 x 5 micron sampling and an entire 96 well or 384g g

Z’-values were comparable between 96 and 384 formats10FI

1520

FI

The image acquisition was done at 5 x 5 micron sampling and an entire 96-well or 384- Z values were comparable between 96 and 384 formats.D it diff t iti iti f I C t d S t M ® M5 i hibiti t t f h 10

15

well plate was scanned in < 5 minutes 1 101 10Despite different sensitivities of IsoCyte and SpectraMax® M5, inhibition constants for each 510

well plate was scanned in < 5 minutes. 1e101e10

CD31 CD311e101e10

CD31 CD31p y p ,

compound could be accurately determined5

The SpectraMax® M5 plate reader was used with 488 nm excitation and emission at CD31 CD31CD31 CD31 compound could be accurately determined.00The SpectraMax M5 plate reader was used with 488 nm excitation and emission at 530nm (c t off 515nm) fo Ale a Fl o ® 488 labeled cell detection and 590nm (c t off no AB Alexa no stim Alexa stimnoAB PE no stim PE stim530nm (cut-off 515nm) for Alexa Fluor® 488 labeled cell detection and 590nm (cut-off 5e95e9 5e95e9

no AB Alexa no stim Alexa stimnoAB PE no stim PE stim

550nm) for Phycoerythrin (PE)-labeled detection 5e9

AG1265e9

AG126550nm) for Phycoerythrin (PE) labeled detection SB202190 AG126SB202190 AG126 Summary0SB202190 G 6

0SB202190 G 6 Summary

0 1 1 10 100 100000.01 0.1 1 10 100

0

0 1 1 10 100 100000.01 0.1 1 10 100

0

Inhibition of VCAMI hibiti f VCAM

u a y0.1 1 10 100 1000

SB202190, uMI hibiti f VCAM HLADR APC0.1 1 10 100 1000

SB202190, uMI hibiti f VCAM HLADR APCInhibition of VCAMInhibition of VCAM •We demonstrated that measurement of adhesion molecules by directly AG126, uMInhibition of HLA-DRInhibition of VCAMVCAM-PE HLADR-APCAG126, uMInhibition of HLA-DRInhibition of VCAMVCAM-PE HLADR-APC45

•We demonstrated that measurement of adhesion molecules by directly VCAM PE HLADR APCVCAM PE HLADR APC45IsoCyte SpectraMax®M5 labeled antibodies provides a powerful assay for testing efficacy of anti-1.2e9

AG1261.2e9

AG1264e9 IsoCyte SpectraMax M5 labeled antibodies provides a powerful assay for testing efficacy of anti-

AG1264e9 AG126AG1264e9 AG12635 inflammatory compounds1e9

G 6AG1264e9 AG126 1e9

G 6AG1264e9 AG12635

3e9inflammatory compoundsAG126 1e9

IC 13 2AG1261e9

IC 13 2AG1263e9

W h t t d i f l i fl ti k VCAM EAG126AG126

IC50 20 1uMSB202190IC50 13.23e9 IC50 8.8 uM IC50 20 1uMSB202190IC50 13.23e9 IC50 8.8 uM25 •We have tested expression of several inflammation markers: VCAM, E-AG126

IC50 8 9 uMSB202190 8e8 IC50 20.1uMSB2021903e9 8e8 IC50 20.1uMSB2021903e9252e9

We have tested expression of several inflammation markers: VCAM, El ti HLA DR ll CD31 HUVEC ti l t d ith i fl ti IC50 8 8

IC50 8.9 uMSB

SB202190

selectin, HLA-DR, as well as CD31 on HUVEC stimulated with inflammation IC50 8.8SB202190IC50 8 25 uM

6e8IC50 13 02e9 SB6e8IC50 13 02e9 SB

151 9

selectin, HLA DR, as well as CD31 on HUVEC stimulated with inflammation t ki ith ith t ti i fl t dIC50 8 5 M

IC50 8.25 uM IC50 13.02e9 SB202190IC50 13.02e9 SB2021901e9 cytokines, with or without anti-inflammatory drugsIC50 8.5 uM

4e8 SB202190C 4e8 SB202190Ccytokines, with or without anti inflammatory drugs

4e8 SB2021901 9

IC50 8.9 uM 4e8 SB2021901 9

IC50 8.9 uM0 1 1 10 100 1000

50 •We show that up-regulation and inhibition of inflammation markers can be Non stim lated Cells stimulated 1e9 IC50 15 2uM1e9 IC50 15 2uM0.1 1 10 100 10000.1 1 10 100 1000 •We show that up-regulation and inhibition of inflammation markers can be Non stimulated ll

Cells stimulated ith c tokines

I fl ti A2e8

IC50 15.2uM2e8

IC50 15.2uMConcentration, UMConcentration UM efficiently determined by IsoCyte or SpectraMax® M5 in a multiplexed cells with cytokines

Inflammation Assay00

Concentration, UM efficiently determined by IsoCyte or SpectraMax® M5 in a multiplexed yHUVEC cells (Lonza) were stimulated Figure 2. HUVEC cells were stimulated with cytokines for 24h Fluorescent 00 1 1 10 100 1000

000 1 1 10 100 1000

0assay formatHUVEC cells (Lonza) were stimulated Figure 2. HUVEC cells were stimulated with cytokines for 24h. Fluorescent

i t iti d b S t M ® M5 I hibiti t t f 0.01 0.1 1 10 100 100000.1 1 10 100 10000.01 0.1 1 10 100 100000.1 1 10 100 1000 assay format

with inflammatory cytokines (TNF, intensities were measured by SpectraMax® M5. Inhibition constants for Concentration uMConcentration UMConcentration uM Concentration uMConcentration uMConcentration UMConcentration uM Concentration uMy

D t t d f th k ti i fl t d AG126 with inflammatory cytokines (TNF, IFN R&D) for 24h Cells stained with

y pdifferent compounds could be determined by either IsoCyte or SpectraMax®

Concentration, uMConcentration, UMConcentration, uM Concentration, uMConcentration, uMConcentration, UMConcentration, uM Concentration, uM −Data generated for three known anti-inflammatory compounds: AG126, EIFNR&D) for 24h. Cells stained with different compounds could be determined by either IsoCyte or SpectraMax® Data generated for three known anti inflammatory compounds: AG126, SB202190 d PDTC fi tilit f th f t ti ti

PE

directly conjugated antibodies (Becton M5 instruments. SB202190 and PDTC confirms utility of the assay for testing anti-M-

directly conjugated antibodies (Becton Di ki ) i t dh i l l

M5 instruments. SB202190 and PDTC confirms utility of the assay for testing antii fl l lA

M

Dickinson) against adhesion molecules Figure 4 Top Multiplexing : Inflammation plus toxicity assay inflammatory moleculesCA) g

as recommended by the BD protocol Figure 4. Top. Multiplexing : Inflammation plus toxicity assayT i ki i hibit AG126 d 38ki i hibit SB202190 b th d

inflammatory moleculesVCas recommended by the BD protocol. S l d ib di d f

Tyrosine kinase inhibitor AG126 and p38kinase inhibitor SB202190 both decrease •Multiplexed inflammation assay can be used for testing efficacy of anti•Plavec I Sirenko O Privat S Wang Y Dajee M Melrose J Nakao B Berg EL Butcher EC Method for analyzing signaling networks in complex cellular systems PNASSelected antibodies used for y p

up-regulation of VCAM Dose-dependent inhibition of the expression of VCAM is •Multiplexed inflammation assay can be used for testing efficacy of anti-•Plavec I, Sirenko O, Privat S, Wang Y, Dajee M, Melrose J, Nakao B, Berg EL, Butcher EC. Method for analyzing signaling networks in complex cellular systems. PNAS 2004;101(5):1223-8.

optimization:up-regulation of VCAM. Dose-dependent inhibition of the expression of VCAM is

d b fl f k

ginflammatory compounds in either 96 or 384 multi well formats in a high

2004;101(5):1223 8.•Neish AS, Williams AJ, Palmer HJ, Whitley MZ and T Collins. Functional analysis of the human vascular cell adhesion molecule 1 promoter. JEM, 176, 1583-1593optimization: measured by fluorescence on IsoCyte. Decrease of CD31 marker suggests toxicity inflammatory compounds in either 96 or 384 multi-well formats in a high-a

e s S, a s J, a e J, t ey a d Co s u ct o a a a ys s o t e u a ascu a ce ad es o o ecu e p o ote J , 6, 583 593•Michael F. Iademarco SB, Jay J. McQuillan, Glenn D. Roseny, and Douglas C. Dean Characterization of the Promoter for (VCAM- 1)* JBC 1992 267, 16323-16329

•PE-conjugated anti-CD106 (VCAM), easu ed by uo esce ce o soCyte ec ease o C 3 a e suggests to c ty

effect of AG126 at high dosesy p g

throughput compatible formatexa , y , y, g ( ) ,

•Jensen LE, Whitehead AS. ELAM-1/E-selectin promoter contains an inducible AP-1/CREB site and is not NF-kappa B-specific. Biotechniques. 2003 Jul;35(1):54-6, 58.PE conjugated anti CD106 (VCAM), •APC conjugated anti E selectin

effect of AG126 at high doses. throughput compatible format

Ale

p pp p q ( )•Traenckner E B, S Wilk, and P A Baeuerle. A proteasome inhibitor prevents activation of NFkB and stabilizes a newly phosphorylated form of IkA that is still bound to NF-•APC-conjugated anti-E-selectin,

g p p

-A kappa B. EMBO J. 1994 13(22): 5433–5441B h lt KE K t RS R tl d JC Ad i bl k IFN i d d h h l ti f STAT1 S 727 t d h ti ti JI 2009 183(10) 6767 77•APC-conjugated anti-HLA-DR Bottom Multiplexing: Different inflammation pathways31 •Barnholt KE, Kota RS, Rutledge JC. Adenosine blocks IFN-g-induced phosphorylation of STAT1 on Ser 727 to reduce macrophage activation. JI 2009 183(10):6767-77Pl t i LC M h i f t I d t II i t f di t d i lli N t R I l 2005(5) 375 86•APC conjugated anti HLA DR,

AF488 j t d ti CD31(PECAM)Bottom. Multiplexing: Different inflammation pathwaysD

3 •Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol. 2005(5):375-86.Baron M and Davignon JL Inhibition of IFN Induced STAT1 Tyrosine Phosphorylation by Human CMV Is Mediated by SHP2 The JI 2008 181 5530 5536•AF488-conjugated anti-CD31(PECAM) VCAM and HLA-DR are under control of different signaling pathways: NFkB and C

D •Baron M and Davignon JL Inhibition of IFN--Induced STAT1 Tyrosine Phosphorylation by Human CMV Is Mediated by SHP2. The JI, 2008, 181, 5530 -5536 j g ( ) VCAM and HLA DR are under control of different signaling pathways: NFkB and J k St t ti l B th i fl ti k d b I C t i Jak-Stat respectively. Both inflammation markers were measured by IsoCyte in p y y ythe same assay p38Kinase inhibitor SB202190 and tyrosine kinase inhibitor FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. Molecular Devices, the Molecular Devices logo, and all other trademarks, unless otherwise indicated, are the property of Molecular Devices, Inc. ©2010 Molecular Devices, Inc. the same assay. p38Kinase inhibitor SB202190 and tyrosine kinase inhibitor

2 d l f dAG126 decrease up-regulation of VCAM and HLA-DR.p g