Key Variables in ICS Assays

Holden T. Maecker

The original BD ICS assay protocol

CD

69 P

E

anti-TNF FITC

Centrifuge, aliquot intostaining tubes or freeze

+ FACSLyse,10 min+ EDTA/PBS

+ BrefeldinA

1 ml whole blood +Ag + CD28 + CD49d

2h 4h 15 min

FACSPerm, 10 min

mAb staining,30-60 min

3- or 4-colorFlow cytometry

Wash Wash, fix,analyze

6h @ 37oC, EDTA, FACS Lyse, FACS Perm 2

PBMC or whole blood

to flow cytometer

Plate with lyophilized Ag+BFA

Plate with lyophilized Ab

96-well plate ICS protocol with lyophilized reagents

What are the key variables?

• Stimulation• PBMC vs. whole blood• Resting PBMC before activation• Activation/staining in plates vs. tubes• Costimulatory Abs (CD28+CD49d)• Stimulation time• Brefeldin A and/or monensin

• Processing and staining• Use of EDTA or not• Method of fixation/permeabilization• Freezing of cells post-fixation• Surface vs. intracellular staining for CD3, CD4, CD8, etc.• Fluorochromes and fixation (APC-Cy7, AmCyan)• Staining time, temperature, and agitation• Number of washes (post-fix and post-stain)

• Acquisition and analysis• Number of events to collect• Gating• Interpretation of results, precision, comparisons with other assays

Effect of overnight rest on cryo PBMC

MFI of IFN+

100 10000 10000000

250

500

750

Reciprocal Dilution

No rest, 6 h peptide stimulation

Overnight rest, 6 h peptide stimulation

% IFN+

100 10000 10000000

1

2

3

Reciprocal Dilution

Correlation of tube vs. plate ICS

0 1 2 3 40

1

2

3

4

r2 = 0.73p = 0.0002slope = 1.1

r2 = 0.96p < 0.0001slope = 1.2

0 10 20 300

10

20

30

r2 = 0.79p < 0.0001slope = 0.84

0 10 200

10

20

r2 = 0.74p = 0.0002slope = 0.78

tube

B. 96-well PBMCA. 96-well whole blood

CD8

0.0 0.5 1.0 1.5 2.0 2.50.0

0.5

1.0

1.5

2.0

2.5

r2=0.87p<0.0001slope 0.88

0.0 2.5 5.0 7.5 10.00.0

2.5

5.0

7.5

10.0

r2= 0.94p<0.0001slope 0.91

0 2 4 6 8 10 12 140

2

4

68

10

1214

r2=0.95p<0.0001slope 0.93

CD4

0.000.250.500.751.001.250.00

0.25

0.50

0.75

1.00

1.25

r2= 0.95p<0.0001slope 0.94

Peptidemix

SEB

plate

CD8

0 1 2 3 40

1

2

3

4

CD4

% IF

N+

cel

ls%

IFN+

cel

ls

% IFN+ cells% IFN+ cells % IFN+ cells% IFN+ cells

Similar IFN MFI in tubes vs. plates

CD4 CD80

100

200

300

CD4 CD80

100

200

300 tube

plate

CD4 CD80

100

200

300

B. 96-well PBMCA. 96-well whole blood C. 24-well whole blood (HIV+)

IFN

me

an f

luo

resc

enc

e

Cell recovery in tubes vs. plates

0

25

50

75

100

Whole Blood PBMC

Tub

e

96-w

ell

24-w

ell

Tub

e

96-w

ell

% C

D3+

cel

l rec

over

y

Effect of costimulatory Abs (CD28+49d)

frozen fresh

0

25

50

75

100

125

IFNg

IL-2

pp65 peptide mix

% o

f co

stim

ula

ted

resp

on

se

frozen fresh

0

25

50

75

100

125

SEB

Monensin vs. Brefeldin Summary

• Brefeldin A preferred (10 g/mL):• TNF, IFN, IL-2, IL-4, IL-12

• When combining any of the above with CD107, TGF, or IL-10:• 5 g/mL monensin + 5 g/mL brefeldin A

What are the key variables?

• Stimulation• PBMC vs. whole blood• Resting PBMC before activation• Activation/staining in plates vs. tubes• Costimulatory Abs (CD28+CD49d)• Stimulation time• Brefeldin A and/or monensin

• Processing and staining• Use of EDTA or not• Method of fixation/permeabilization• Freezing of cells post-fixation• Surface vs. intracellular staining for CD3, CD4, CD8, etc.• Fluorochromes and fixation (APC-Cy7, AmCyan)• Staining time, temperature, and agitation• Number of washes (post-fix and post-stain)

• Acquisition and analysis• Number of events to collect• Gating• Interpretation of results, precision, comparisons with other assays

EDTA addition in plates and tubes

Tu

be

s n

o E

DT

A

Tu

be

s E

DT

A

Pla

tes

no

ED

TA

Pla

tes

ED

TA

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

% C

D3+

CD

8+ IF

N +

mea

n of

trip

licat

e sa

mpl

es

Fix/perm comparison: monocyte IL-12

Fix/perm comparison: T cell TGF

TG

F

PE

TG

F

PE

TG

F

PE

TG

F

PE

BD FACS Lysing Solution BD Cytofix/CytopermBD FACS Permeabilizing Sol’n 2 BD Perm Wash

TGF staining and activation mediaT

GF

PE

TG

F

PE

TG

F

PE

Freezing activated cells in FACS Lyse

Surface vs. IC staining for CD3

surface stain intracellular stain

CD3 Alexa 700Clone SP34-2 @0.8g/test

SEB stimulated PBMCs

Surface vs. IC staining for CD4 & CD8

surface stain intracellular stainSEB stimulated PBMCs

CD8 Qdot 605 @ 0.25ul CD4 AmCyan @ 60ng

CD8 APC-hilyte 750 @125ngCD4 AmCyan @ 60ng

CD8+ IFN+ CD8+ IFN+ IL-2+ CD4+ IFN+ CD4+ IFN+ IL-2+

A

B

C

CD3 Pacific Blue

Sid

e Li

ght S

catte

r

CD4 AmCyan

IL-2 PE IL-2 PE

CD

8 A

PC

-Cy7

IFN

FIT

C

IFN

FIT

C

Sid

e Li

ght S

catte

r

Sid

e Li

ght S

catte

r

Sid

e Li

ght S

catte

r

Sid

e Li

ght S

catte

r

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5

All events CD3+ gate

CD4- CD8+ gate CD4+ CD8- gate

CD27 APC CD27 APC CD27 APC CD27 APC

Degradation of APC-Cy7 on stained cells

0

25

50

75

100

PBS/BSA

PBS/formaldehyde

nostorage

overnight4oC

overnightroom temp

% s

pil

love

rA

PC

-Cy7

->

AP

C

In BDSF

BDSF and AmCyan don’t mix

AmCyan Signal:Noise

-0.5 0.0 0.5 1.0 1.5 2.0 2.510.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

PBS/BSA

1% PFABDSF

hours

Sig

nal

:No

ise

FITC Spillover

-0.5 0.0 0.5 1.0 1.5 2.0 2.50

25

50

75

100

hours

Sp

illo

ver

Am

Cya

n -

>F

ITC

IC staining time, volume, and mixing

0

20

40

60

80

100

120

140

160

50 100 150 200 250 300 350

volume, ul

IFN

FIT

C M

FI

30 min

60 min

30 min w/mix

60 min w/mix

What are the key variables?

• Stimulation• PBMC vs. whole blood• Resting PBMC before activation• Activation/staining in plates vs. tubes• Costimulatory Abs (CD28+CD49d)• Stimulation time• Brefeldin A and/or monensin

• Processing and staining• Use of EDTA or not• Method of fixation/permeabilization• Freezing of cells post-fixation• Surface vs. intracellular staining for CD3, CD4, CD8, etc.• Fluorochromes and fixation (APC-Cy7, AmCyan)• Staining time, temperature, and agitation• Number of washes (post-fix and post-stain)

• Acquisition and analysis• Number of events to collect• Gating• Interpretation of results, precision, comparisons with other assays

Number of events to acquire

CALCULATING SAMPLE SIZE FOR RARE EVENT ANALYSIS

Enter the estimated values in the grey fields:

% bkgd= 0.1

p(av)= 0.0015

%pos= 0.2delta= 0.001

       For power of 90%, p<0.05, N= 25761For power of 99%, p<0.005, N= 71592For power of 80%, p<0.05, N= 18572

_______________________________________________________________

________# events to collect__________

% bkgdlowest % positive

90% power, p<0.05

99% power, p<0.005

_______________________________________________________________0.01 0.02   260,000 720,0000.01 0.05   32,000 90,0000.01 0.1   12,000 32,0000.02 0.05   67,000 190,0000.02 0.1   16,000 45,0000.03 0.05   170,000 480,0000.03 0.1   23,000 63,0000.04 0.1   33,000 93,0000.05 0.1   52,000 140,0000.06 0.1   86,000 240,0000.07 0.1   160,000 450,0000.08 0.2   17,000 46,000

0.1 0.2   26,000 72,000_______________________________________________________________

Importance of gating dim cells

Multi-site lyophilized control cell data

Individual (Manual) Analysis

Coclta

il 1 C

D4

Cockta

il 1 C

D8

Cockta

il 2 C

D4

Cockta

il 3 C

D80

10

20

30

32% 29% 7% 14%

% c

ytok

ine

+ c

ells

Central (Automated) Analysis

Coclta

il 1 C

D4

Cockta

il 1 C

D8

Cockta

il 2 C

D4

Cockta

il 3 C

D80

10

20

30

7% 3% 3% 3%

% c

ytok

ine

+ c

ells

Gating

CD

8 P

erC

P-C

y5.5

Sid

e S

catt

er

CD

69

PE

Sid

e S

catt

er

0 200 400 600 800 1000Forward Scatter

R1

100 101 102 103 104

CD3 APC

R3

100 101 102 103 104

IFN FITC

R4

100 101 102 103 104

CD3 APC

R2

Manual analysis

0.55%

ungated ungated

gated on R1 gated onR1&R2&R3

CD

8 P

erC

P-C

y5.5

Sid

e S

catt

er

CD

69

PE

Sid

e S

catt

er

0 200 400 600 800 1000Forward Scatter

R1

100 101 102 103 104

CD3 APC

R4

R5

100 101 102 103 104

R3R2

CD3 APC

10 0 10 1 10 2 10 3 10 4

IFN FITC

R7

R6

ungated gated on R1

ungated gated onR1&R3&R5

0.56%

Batch analysis with dynamic gates

What are the key variables?

• Stimulation• PBMC vs. whole blood• Resting PBMC before activation• Activation/staining in plates vs. tubes• Costimulatory Abs (CD28+CD49d)• Stimulation time• Brefeldin A and/or monensin

• Processing and staining• Use of EDTA or not• Method of fixation/permeabilization• Freezing of cells post-fixation• Surface vs. intracellular staining for CD3, CD4, CD8, etc.• Fluorochromes and fixation (APC-Cy7, AmCyan)• Staining time, temperature, and agitation• Number of washes (post-fix and post-stain)

• Acquisition and analysis• Number of events to collect• Gating• Interpretation of results, precision, comparisons with other assays

Holden’s rules for ICS (and life)

• Validate your assay, but don’t validate that the earth is round• take advantage of others’ data if it’s sound

• Know the inherent variability in your assay• don’t talk about significant differences unless you

run at least triplicate samples

• Avoid comparing apples and oranges• any changes to the assay can produce variability,

so keep protocols constant and run samples in parallel whenever possible

Acknowledgements

• Laurel Nomura• Smita Ghanekar• Maria Jaimes• Margaret Inokuma• Sonny Bhatia• Joyce Ruitenberg• Maria Suni• Jack Dunne• Skip Maino

• Brinda Emu• Rebecca Hoh• Steve Deeks• Jeff Martin• Mike McCune• Doug Nixon