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MULTIPARAMETER IMMUNOFLUORESCENCE
Carleton C. Stewart, Ph.D. & Sigrid J. Stewart
R LLOSWEARKP
L Cancer Instituteaboratory
Flowof
CytometryDepartment of Health, State of New York
Elm and Carlton Streets
Buffalo, New York 14263
phone: 716-648-5480
fax: 716-648-8806
email: [email protected]
ANTIBODY BINDING TO CELLS
RPCI
LFC
Kf
Kr
Ab + Ep AbEp
Kf range is usually ~ 106
Kr range is usually ~ 10-3
Ka = Kf/Kr = 106/10-3 = 109
Kf =AbEpAb
EpKr = AbEp
Ab
Ep
THE LAW OF MASS ACTION
Specific:Fab to epitopeFc to Fc receptorbinding is high affinity and saturable
Non Specific:binding is low affinity and not saturable
WAYS ANTIBODIES BIND TO CELLS
Specific Activity is the concentration
of bindable antibody to its epitope
divided by the protein concentration.
2
[F(ab') ] SA =
(protein)
Reasons Antibodies do not bind to cells:
•overconjugation•not purified•degradation of binding site•aggregation
STORING OF ANTIBODIES :
Proteases destroy antibodies in:• ascitic fluid• serum• bacteria
Use sodium azide
Use highly purified albumin or gelatin as carrier
Purify antibodies immediately
TITERING ANTIBODIESRPCI
LFC
SPECIFIC ANTIBODY
NON-SPECIFIC ANTIBODY
CONCENTRATION
AM
OU
NT B
OU
ND
3
3 µgs/n = 2.5
1 µgs/n = 2.1
0.3 µgs/n = 2.4
0.1 µgs/n = 4.1
0.03 µgs/n = 4.8
0.01 µgs/n = 4.6
0.003 µgs/n = 3.5
0.001 µgs/n = 3.2
auto
8765432102
3
4
5
Dilution
Sig
nal to
Nois
eTITER
101 102 103 104101 102 103 104101 102 103 104
1 µg S/NAb 278IC 5.8
isotypecontrol
antibody
cytokeratin
.3 µg S/NAb 100IC 3.6
101 102 103 104
.01 µg S/NAb 25.7IC 2.6
nu
mb
er
65432100
10
20
30
40
50
60
Dilution
Sig
nal to
Nois
e
TITER
10 410 4 10 1 10 2 10 3 10 410 1 10 2 10 3 10 4
PE-CD4 TC-CD810 1 10 2 10 310 1 10 2 10 3
FITC-CD3
nu
mb
er
10 1 10 2 10 3 10 410 1 10 2 10 3 10 4
VERIFICATION OF ANTIBODY COMBINATIONS
101 102 103 104
10
1
10
2
10
3
10
4
R1
PE
-CD
4
FITC-CD3
x = 69y = 94
PE
- C
D4
101 102 103 104
10
1
10
2
10
3
10
4
R2
TC-CD8
x = 98
NEW BATCH
R1
101 102 103 104
10
1
10
2
10
3
10
4
PE
-CD
4
FITC-CD3
x = 62 faily = 96 pass R2
PE
- C
D4
TC-CD81 2 3 4
10
1
10
2
10
3
10
4
10 10 10 10
x = 97 pass
CONTROL BATCHCONTROL BATCH
BLOCKING IS IMPORTANT
INDIRECT IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody: Second Antibody:murine monoclonal fluoresceinatedantibody goat anti-mouse
IgG F(ab')2Fab
epitope
FcR
A B C D
Fc
ISOTYPE CONTROL- myeloma protein
AUTOFLUORESCENCE CONTROL
E F G H
BLOCKING WITH GOAT IgG
goat IgG
add MabFab
add fluoresceinated goat anti-mouse IgG F(ab')2
VERIFICATION OF BLOCK
1. FcR and non-specific binding
FL-MAB + PE-mIgG
2. gIgG + FL-MAB + PE-mIgG
EFFECT OF BLOCKING ON ANTIBODY BINDING TO MONONUCLEAR CELLS
LO
G F
LU
OR
ES
CEN
CE
CELL VOLUME
nu
mb
er
TOTALA
B
C
D
channel number
variation in gamma 1 myeloma protein binding to macrophages
0
510
1520
25
3035
4045
50
0 17 39 4 23 13 21
mopc myeloma protein
Second Reagent Qualitylo
g fl
uore
scen
ce
cell volume
F(ab’)2 of anti IgG anti IgG
DEAD CELLS CAN BE A PROBLEM
• They bind antibodies non-specifically• They masquerade as specific subsets• They cause data misinterpretation
ANTIBODIES BIND NON-SPECIFICALLY TO DEAD CELLS
FL-KAPPA
PE-L
AM
BD
A
dead cells
ALL CELLS VIABLE CELLS
A B
lysed, washedcells
+ 5 µg EMA
10 min.
18 cm.
EMA PROCEDURE
WASH, FIX, AND ANALYZE
1
3
2
Evaluating Viability with Ethidium Monoazide
FSC
SS
C
10 20 30 40 50 60 70 80 90 100 120
Forward Scatter Height -->
10
20
30
40
50
60
70
80
90
100
120
Sid
e S
catte
r Heig
ht -->
R9
FSC
SS
C
10 20 30 40 50 60 70 80 90 100 120
Forward Scatter Height -->
10
110
210
310
4
Fluore
scence
Thre
e H
eig
ht -->
R2
FSC
FL3-E
MA
10 20 30 40 50 60 70 80 90 100 120
Forward Scatter Height -->
10
20
30
40
50
60
70
80
90
100
120
Sid
e S
catte
r Heig
ht -->
R9R9
% dead = 5%
R2
% dead in gate = 1%
R9
ONE COLOR IMMUNOPHENOTYPING
RPCI
LFC
1. IgG BlockMAB wash FL-second antibody F(ab')2 wash
2. IgG BlockB-MAB wash FL-Avidin wash
3. IgG BlockFL-MAB wash
SINGLE COLOR IMMUNOPHENOTYPING
CORRELATED (LIST MODE) DATA ACQUISITION
Entry No. Value Cell Number Parameter1 80 1 FSC2 100 1 SSC3 40 1 Green4 20 1 Red5 90 2 FSC6 120 2 SSC7 100 2 Green8 110 2 Red- 50 n FSC- 75 n SSC- 110 n Greenk 120 n Red
Analysis of List Mode Data
CD4 FLUORESCENCE
nu
mb
er
of
cells
forward scatter CD4 fluorescence
A
B
C
region A
region B region C
STRATEGIES IN MULTICOLOR FLOW CYTOMETRY
RPCI
LFC
CELLULAR ANTIGENS
AdhesionReceptors
Metabolic
cytokines
structureenzymes
courtesy of Jim Bender
FLOW CYTOMETRY VS MICROSCOPIC IMAGING
so rt ing c e llsm o le c ular quant it at io nant ige n c o e xpre ss io nrare c e lls de t e c t io n
c e llu lar int e rac t io nspac ial re lat io nshipsc o m part m e nt alizat io ns ignal t o no ise re je c t io n
TWO COLOR IMMUNOPHENOTYPING
Antibodies labeled with fluorescein
Antibodies labeled with phycoerythrin
RPCI
LFC
TWO COLOR IMMUNOPHENOTYPING
1. IgG Block + MAB wash FL- second antibody F(ab')2 wash
IgG Block + PE-MAB wash
2. IgG Block + B-MAB + FL-MAB wash PE-Avidin wash
3. IgG Block + FL-MAB + PE- MAB wash
COMBINED INDIRECT AND DIRECT
IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody: mMAB Second Antibody: FGAM PE-mMAB
NO BLOCKC
D8 +
FG
AM
PE-CD4
21%
6%
49%
VERIFICATION OF BLOCK
• Second Reagent Block
gIgG + MAB wash FL-GAM wash PE-mIgG
gIgG + MAB wash FL-GAM wash mIgG + PE-mIgG
COMBINED INDIRECT AND DIRECT IMMUNOFLUORESCENCE STAINING
BLOCKING
Primary Antibody: mMAB Second Antibody: FGAM PE-mMAB
BLOCKEDC
D8 +
FG
AM
PE-CD4
42%
12%
THREE COLOR IMMUNOPHENOTYPING
Antibodies labeled with Fluorescein
Antibodies labeled with Phycoerythrin
Antibodies labeled with Tandem Complex to Avidin
Tandem Complexes are Texas Red or CY 5 coupled to Phycoerythrin
Per CP is a natural Tandem Complex of peridinin and chlorophyll a protein
SINGLE COLOR HISTOGRAMS
TWO COLOR PATTERN
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD
4 -
->
FL1-CD3
FL2-C
D4 color
CD3-CD4- black
CD3+CD4- blue
CD3-CD4+ cyan
CD3+CD4+green
THREE COLOR PATTERN
CD3C
D4
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD
4 -
->
CD3
CD
4
CD8C
D8
10 1 10 2 10 3 10 4
CD8 -->
101
102
103
104
CD
4 -
->10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD
8 -
->
FOUR COLOR SINGLE LASER IMMUNOPHENOTYPING
Antibodies labeled with fluorescein
Antibodies labeled with phycoerythrin (PE)
Antibodies labeled with PE/Texas Red
Antibodies labeled with PE/CY5 or PerCP
FOUR COLOR DUAL LASER IMMUNOPHENOTYPING
Antibodies labeled with fluorescein
Antibodies labeled with phycoerythrin (PE)
Antibodies labeled with PE/CY5 or PerCP
Antibodies labeled with APC, CY5 or CY7
10 1 10 2 10 3 10 4
CD56 -->
101
102
103
104
CD
4 -
->
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD
4 --
>
CD3CD3 CD310 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD
56 -
->
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD
8 -
->
CD5610 1 10 2 10 3 10 4
CD56 -->
101
102
103
104
CD
8 --
>
CD56 CD810 1 10 2 10 3 10 4
CD8 -->
101
102
103
104
CD
4 -
->
FOUR COLOR PATTERN
CD
4
CD
8
CD
56
CD
8
CD
4
CD
4
FL1 FL3 FL3
FL2
FL2
FL1
FL1
FL1
FL2
FL2
FL3
FL3
VISUALIZING EACH POPULATION
BIVARIATE DOT PLOTS CAN BE USED TO DISPLAY THE PATTERNS GENERATED BY MULTIPARAMETER DATA.
FL1
FL4
FL2
FL4
FL3
FL4
FL1
FL3
FL2
FL3
FL1
FL2
4 COLOR
3 COLOR
2 COLOR
UNDERSTANDING BINARY LOGIC IS USEFUL
Note that in binary logic the 1st parameter is sequenced as -/+, two parameters as --/++, 3 parameters as ----/++++, etc.. for as many parameters that are measured. The problem encountered after 3 parameters is...how does one visualize the multiple populations?
P1 P2 P3 P4- - - -+ - - - P1- + - -+ + - - P2- - + -+ - + -- + + -+ + + - P3- - - ++ - - +- + - ++ + - +- - + ++ - + +- + + ++ + + + P4
Ab2
Ab1 Ab3 Ab3
Ab2
Ab1
Ab1 Ab2 Ab3 color Ab1 Ab2 Ab3 colorneeg.(-) neg.(-) neg.(-) grey neg.(-) neg.(-) pos.(+) rustos.(+) neg.(-) neg.(-) yellow pos.(+) neg.(-) pos.(+) blueneg.(-) pos.(+) neg.(-) cyan neg.(-) pos.(+) pos.(+) violetnos.(+) pos.(+) neg.(-) green pos.(+) pos.(+) pos.(+) red
COLOR PATTERNS USED TO VIEW COEXPRESSION
R1 R2 R3 R4 boolean colorAb1 Ab2 Ab3 Ab4
- - - - not(R1 or R2 or R3 or R4 ) grey+ - - - R1 and not (R2 or R3 or R4 ) ye llow- + - - R2 and not (R1 or R3 or R4 ) cyan+ + - - R1 and R2 and not (R3 or R4 ) green- - + - R3 and not (R1 or R2 or R4 ) rust+ - + - R1 and R3 and not (R2 or R4 ) blue- + + - R2 and R3 and not (R1 or R4 ) v io le t+ + + - R1 and R2 and R3 and not R4 red- - - + R4 and not (R1 or R2 or R3 ) grey+ - - + R4 and R1 and not (R2 or R3 ) ye llow- + - + R4 and R2 and not (R1 or R3 ) cyan+ + - + R4 and R1 and R2 and not R3 green- - + + R4 and R3 and not (R1 or R2 ) rust+ - + + R4 and R1 and R3 and not R2 blue- + + + R4 and R2 and R3 and not R1 vio le t+ + + + R4 and R2 and R3 and R1 red
Ab4 is t he gat ing ant ibody
Boolean Logic Table For Four Color Immunophenotyping
A B C
D E F
Ab1 Ab1 Ab1
Ab1 Ab1 Ab1
Ab
2A
b2
Ab
2A
b2
Ab
2A
b2
Types of Antigen Expression
10 1 10 2 10 3 10 4
CD7 -->
10
1
10
2
10
3
10
4
CD19 -->
1 2
3 410 1 10 2 10 3 10 4
CD2 -->
10
1
10
2
10
3
10
4
CD19 -->
18 19
20 21
10 1 10 2 10 3 10 4
CD2 -->
10
1
10
2
10
3
10
4
CD7 -->
5 6
7 810 1 10 2 10 3 10 4
CD7 -->
10
1
10
2
10
3
10
4
CD19 -->R1
1 2
3 4
A B
DC
CD19
CD19
CD19
CD2
CD7
CD7
CD2
CD7
INTERPRETING COEXPRESSION
THREE OR MORE COLOR IMMUNOPHENOTYPING
1. IgG Block + MAB wash Biotin- second antibody F(ab')2
wash IgG Block + FL- MAB + PE-MAB + TC- Avidin wash
2. IgG Block + FL-MAB + PE-MAB + B-MAB wash TC-Avidin wash
3. IgG Block + FL-MAB + PE-MAB + TC-MAB wash
TC (third color) = PE/TR or PE/CY5 tandem or PerCP
STRATEGY FOR SELECTING FLUOROCHROME:
EPITOPE DENSITY FLUOROCHROMELow phycoerythrin, APClow-intermediate tandem, CY5high fluorescein, PerCP
STAINING SOP50 µl
washed, andblocked*
whole blood orbone marrow
15 min. on ice
lyse,centrifuge,
decant,blot,and
resuspendpellet wash,
fix,and
analyse
*add 10 µl mIg (10 mg/ml) to 1 ml washed whole blood.
Add antibody combination
TANDEM COMPLEX PROPERTIES TO CONSIDER
• monocyte binding PE-CY5
• light sensitivity PE-CY5
• batch variation PE-TR and PE-CY5
Non-specific Binding to Monocytes
A B C
PerCP-CD4 PE-CY5-CD4 FSC
SS
C
CD
25
CD
25
TC-CD45 TC-CD3
Effect of Light Exposure on PE-CY5 Tandem Fluorescence
PE-fl
uore
scen
ce
EFFECT OF BATCH VARIATION
PE-fl
uore
scen
ce
FSC
SS
C10 20 30 40 50 60 70 80 90 100 120
FSC-H -->
1020
3040
5060
7080
90100
120
SS
C-H
-->
CD16
CD
32
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL3-H -->
101
102
103
104
FL
2-H
-->
CD
32
CD6410 1 10 2 10 3 10 4
FL3-H -->
101
102
103
104
FL
1-H
-->
CD
16
CD64
Fc Receptor Expression on Blood Leukocytes
CD16 CD64
All Cells
CD16 CD64
neutrophils
T-cells
eosinophils basophils
monocytes NK cells
B-cells
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL3-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL3-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL3-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL3-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL3-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL3-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL1-H -->
101
102
103
104
FL
2-H
-->
10 1 10 2 10 3 10 4
FL3-H -->
101
102
103
104
FL
2-H
-->
CD
32
CD
32
CD
32
CD
32