r l l oswe ark p l cancer institute aboratory flow of cytometry isac xx tutorial carleton c. stewart...
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R LLOSWEARKP
L Cancer Instituteaboratory
Flowof
Cytometry
ISAC XXISAC XX
TUTORIALTUTORIAL
Carleton C. StewartCarleton C. Stewart
Sigrid J. StewartSigrid J. Stewart
ANTIBODY BINDING TO CELLSANTIBODY BINDING TO CELLS
RPCI
LFC
FcFab
Fab
Light ChainKappa or Lambda
IgG1IgG2aIgG2bIgG2cIgG3
IgM
IgA
IgD
IgE
ANTIBODY STRUCTURE
1000 EPITOPES/ANTIGEN
2 LIGHT CHAINS10 HEAVY CHAINS
20,000 CLONES
Heavy Chain
ss
ss
ss
papain pepsin
ss
Kf range is usually ~ 106
Kr range is usually ~ 10-3
Ka = Kf/Kr = 106/10-3 = 109
Kf
Kr
Ab + Ep AbEp
THE LAW OF MASS ACTIONTHE LAW OF MASS ACTION
X[Ab] [Ep]RATE f = Kf X[AbEp]RATE r = Kr
AT EQUILIBRIUM RATE f = RATE r = 0 and
[AE]=Ka[Ab][Ep]/(1+Ka[Ab])
Specific:Fab to epitope
Fc to Fc receptor
binding is high affinity and saturable
Non Specific:binding is low affinity and not saturable
WAYS ANTIBODIES BIND TO CELLSWAYS ANTIBODIES BIND TO CELLS
RPCI
LFC
Specific Activity is the concentration
of bindable antibody to its epitope
divided by the protein concentration.
2
[F(ab') ] SA =
(protein)
RPCI
LFC
Reasons Antibodies do not bind to cells:
•overconjugation
•not purified
•degradation of binding site
•aggregation
RPCI
LFC
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
RPCI
LFC
1 x 10-11 1 x 10-10 1 x 10-9 1 x 10-8 1 x 10-7
Ka=10Ka=1077
Ka=10Ka=1088
Ka=10Ka=1099
Ka=10Ka=101010
Ka=10Ka=101111
EFFECT OF AFFINITY CONSTANT ON ANTIBODY BINDING
Affinity Constant (L/M)
40
60
80
100
- - - -107 108 109 1010 1011
per
cen
t fr
ee
0.01 0.1 1 10 100 1000
ANTIBODY (g/ml)
1000
10
1
0.1
0.01
0.001
0.0001
MEASURED BINDING
SPECIFIC BINDING
NON-SPECIFIC BINDINGKa = 5 x 104
Ka = 5 x 108
ANTIBODY BINDING TO MEMBRANE ANTIGENSA
MO
UN
T B
OU
ND
(n
g/m
l)
0.001
0.01
0.1
1
10
100
1000
0.001 0.01 0.1 1 10 100 1000
AM
OU
NT
BO
UN
D (
ng/
ml)
MEASURED BINDING
SPECIFIC BINDING
NON-SPECIFIC BINDINGKa = 5 x 104
Ka = 5 x 108
ANTIBODY BINDING TO INTRACELLULAR ANTIGENS
ANTIBODY (g/ml)
1000100
0.0001
0.001
ANTIBODY BINDING TO INTRACELLULAR ANTIGENS
HIGH AFFINITY ANTIBODY
ANTIBODY ( g/ml)
AM
OU
NT
BO
UN
D (
ng
/ml)
10 2
10
1
0.1
0.01
0.001 0.01 0.1 1 10
MEASURED BINDING
SPECIFIC BINDING
NON-SPECIFIC BINDING
Ka = 5 x 10 4
Ka = 5 x 10 9
SPECIFIC AND NON-SPECIFIC ANTIBODY BINDING (MEMBRANE)
Specific Binding
high affinity
Ka = 108 - 1010
for Ka = 5 x 108 saturation1.0 µg/ml (0.1 µg/test)
Non-Specific Binding
low affinity
Ka < 105
for Ka = 5 x 104 saturation90 µg/ml (9 µg/test)
SPECIFIC AND NON-SPECIFIC ANTIBODY BINDING (INTRACELLULAR)
Specific Binding
high affinity
Ka = 108 - 1010
for Ka = 5 x 108 saturation0.1 µg/ml (.01 µg/test)
Non-Specific Binding
low affinity
Ka < 105
for Ka = 5 x 104 saturation90 µg/ml (9 µg/test)
TITERING ANTIBODIESRPCI
LFC
MEMBRANE EPITOPESRPCI
LFC
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
Noi
seTITER
MEMBRANE ANTIGENS
MEMBRANE ANTIGENS
1
1.2
1.4
1.6
1.8
2
2.2
300010003331113712410.50.20.0510.017
ng/test
ratio
titer
non-specific and specific binding
CD4 FITC
NU
MB
ER
OF
CE
LL
S Verification of Specific Binding
INTRACELLULAR EPITOPESRPCI
LFC
• destruction of epitope by denaturation
• loss of antigen by extraction
• masking epitope by cross-linking
• non-specific binding due to low affinity antibody by manufacture
• cross-reacting products by GOD
FACTORS THAT AFFECT DETECTION AFTER FIXATION
• positive target cell lineage for antibody
• genotypically identical negative target cell lineage
• several different surrogate positive target cell lineages
• several different negative target cell lineages
VERIFYING ANTIBODY SPECIFICITY
CELLS
• western blot
• evaluation of Ka
VERIFYING ANTIBODY SPECIFICITY
PROCESS
In the beginning there is a high extracellular antibody concentration.
Antibodies diffuse relatively fast into the cell down the electrochemical gradient and bind specifically and non-
specifically.
BARRIERS TO INTRACELLULAR STAINING
• immobilized epitope decreases antibody binding affinity
• molecular crowding limits diffusion
• fixation induced epitope degradation
• fixation induced epitope obstruction
BARRIERS TO SPECIFIC BINDING
• molecular crowding increases non-specific binding
• NSB reduces effective antibody concentration
• washing efficiency reduced due to slow off-rate
• multiple targets specificity with different epitope affinities (e.g. multiple bands on a western blot)
So, what is the answer? Only one. Antibodies of high affinity so that there is a low free antibody concentration to begin with!
101 102 103 104101 102 103 104101 102 103 104
1 µg MCFAb 278IC 5.8
isotypecontrol
antibody
cytokeratin.3 µg MCFAb 100IC 3.6
101 102 103 104
.01 µg MCFAb 25.7IC 2.6
nu
mb
erINTRACELLULAR ANTIGENSINTRACELLULAR ANTIGENS
65432100
10
20
30
40
50
60
Dilution
Sig
nal
to
Noi
seTITER
INTRACELLULAR ANTIGENS
ANTIBODY BINDINGRPCI
LFC
10 20 30 40 50 60 70 80 90 100 110 120
Side Scatter -->
10
11
02
10
31
04
Anti-T
CR
-alp
ha-b
eta
-1 F
ITC
-->
SIDE SCATTER
10 20 30 40 50 60 70 80 90 100 110 120
Side Scatter -->
10
11
02
10
31
04
Anti-T
CR
-alp
ha-b
eta
-1 F
ITC
-->P
E-CD19
Differing Monoclonal Antibody Epitope Binding
10 1 10 2 10 3 10 4
CD8 TC -->
101
102
103
104
Ant
i-TC
R-g
amm
a-de
lta-1
PE
-->
10 1 10 2 10 3 10 4
CD8 TC -->10
110
210
310
4
Ant
i-TC
R-g
amm
a-de
lta-1
PE
-->
PE-CD69
FITC-CD69
CD3 CD3
Importance of Fluorochrome Intensity
10 1 10 2 10 3 10 4
CD8 TC -->
101
102
103
104
Ant
i-TC
R-g
amm
a-de
lta-1
PE
-->
No BFA
10 1 10 2 10 3 10 4
CD8 TC -->10
110
210
310
4
Ant
i-TC
R-g
amm
a-de
lta-1
PE
-->
With BFA
CD4 CD4
Effect of the Drug Brefeldin A on Membrane CD69 Expression
CD69
CD69
TANDEM DYES
RPCI
LFC
PE
-flu
ores
cenc
eVariation In Compensation For PE-CY5 Reagents
PE
-flu
ores
cen
ce8 hours in dark
8 hours in light
TC-CD45 TC-CD3
Effect Of Light Exposure on PECY5 Tandem Fluorescence
A B C
PerCP-CD4 PECY5-CD4 FSC
SSC
CD25
CD25
PECY5 Binding to Monocytes
BLOCKING IS IMPORTANT
RPCI
LFC
DIRECT IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody:murine monoclonal antibody
A
epitope
FcR
Fab
Fc
Fab
Fc
Fab
Fc
Fab
Fc
Fab
Fc
B C D
%+=75%+=75
DIRECT IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody:murine monoclonal antibody
A
epitope
FcR
Fab
Fc
Fab
Fc
Fab
Fc
Fab
Fc
Fab
Fc
B C D
%+=75%+=75
ISOTYPE CONTROL- myeloma protein
AUTOFLUORESCENCE CONTROL
%+=50%+=50
%+=0%+=0
%+=75%-50=25%%+=75%-50=25%
BLOCKING WITH GOAT IgG
goat IgG
A
epitope
FcR
B C D
Fab
Fc
Fab
Fc
%+=50%+=50
INDIRECT IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody:murine monoclonal antibody
Second Antibody:fluoresceinatedgoat anti-mouse IgG F(ab')2
V
m
mF
VFcR
epitope
Fab
Fc
mm
mF Fab
Fc
m
Fab
Fc
m
m
mF
Fab
Fc
m
m
mF
Fab
Fcm
m
mF
INDIRECT IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody:murine monoclonal antibody
Second Antibody:fluoresceinatedgoat anti-mouse IgG F(ab')2
V
m
mF
VFcR
epitope
Fab
Fc
m
mm
m
m m
mF Fab
Fc
m
Fab
Fc
m
m
mF
Fab
Fc
m
m
mF
Fab
Fcm
m
mF
m
directly labeled primarydirectly labeled primary
INDIRECT IMMUNOFLUORESCENCE STAINING
BLOCKING with mIgG
Primary Antibody:murine monoclonal antibody
Second Antibody:fluoresceinatedgoat anti-mouse IgG F(ab')2
V
m
mF
Fab
Fc
m
VFcR
epitope
Fab
Fc
mm
mF
Fab
Fcm
Fab
Fc
m
Fab
Fc
m
m
mF
m
mF
m
mF
m
m m
m m
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. goat IgG + FL-MAB + PE-mIgG
EFFECT OF BLOCKING ON
ANTIBODY BINDING TO
MONONUCLEAR CELLSL
OG
FL
UO
RE
SC
EN
CE
CELL VOLUME
nu
mb
erTOTAL
A
B
C
D
channel number
variation in gamma 1 myeloma protein binding to macrophages
05
101520253035404550
0 17 39 4 23 13 21
mopc myeloma protein
VIABILITY IS IMPORTANT
RPCI
LFC
ANTIBODIES BIND NON-SPECIFICALLY TO DEAD CELLS
FL-KAPPA
PE
-LA
MB
DA
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
10 20 30 40 50 60 70 80 90 100 110 120
Forward Scatter -->
010
2030
4050
6070
8090
100
110
120
Sid
e S
catte
r --
>
SSC
FSC
% dead in gate = 2%
10 20 30 40 50 60 70 80 90 100 110 120
Forward Scatter -->
01
02
03
04
05
06
07
08
09
01
00
110
12
0
Sid
e S
catt
er
-->
FSC
SSC
10 20 30 40 50 60 70 80 90 100 110 120
Side Scatter -->
10
11
02
10
31
04
Ant
i-TC
R-a
lpha
-bet
a-1
FIT
C -
->
FSC
EMA
% dead = 12%
Ab1
Ab3
Ab1
Ab2
A B
Effect of Dead Cells and Antibody Affinity on Immunophenotyping
COMPENSATION
RPCI
LFC
emission intensity
wavelength
FLBFLA
-/\/
\/\/
\/\
--/
\/\/
\/\
/\-
-/\/
\/\/
\/\
-
-
-
+
+FLB
A %B
B %A
0 %B
FLA
Opamp A
optical filters PMT & preamp
20 %A
Opamp B
-
-
+
+FLB
A %B
B %A
5 %B
FLA
Opamp A
optical filtersPMT & preamp
0 %A
Opamp B
-/\/
\/\/
\/\
-
+ =-/\/
\/\/
\/\-
-/\/
\/\/
\/\-
FLB
FLA
-
-
+
+
A %B
B %A
5 %B
Opamp A
20 %A
Opamp B
+ =
+ =
+ =
Uncompensated vs Compensated
FL1
FL
2
FL1
FL
2COMPENSATION IS INTENSITY
DEPENDENT
uncompensatedpartially
compensated
fully compensated
FL
2
COMPENSATE INSTRUMENT USING STAINED CELLS
1. Adjust PMT voltages using unstained cells
2. Adjust compensation for each fluorochrome
10 10 10 10
FITC-CD3
PE-C
D8
10
TC
-CD
2
FITC-CD3
PE-C
D8
TC-CD22 10 10
10 2 10 4
EC
D-C
D4
FITC-CD310 10 10 10
EC
D-C
D4
TC-CD2
10 2 10 10
PE-C
D8
ECD-CD4
UNCOMPENSATED
SOFTWARE COMPENSATION
101 102 103 104
10
1
10
2
10
3
10
4
PE
CD
8 --
>
PE
-CD
8
TC-CD2101 102 103 104
10
1
10
2
10
3
10
4
PE
-CD
8
ECD-CD4
101 102 103 104
10
1
10
2
10
3
10
4
TC
-CD
2
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
EC
D C
D4
-->
EC
D-C
D4
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
EC
D-C
D4
TC-CD2
101 102 103 104
10
1
10
2
10
3
10
4
PE
-CD
8
FITC-CD3
R1
R2
R3
R4
R5
R6
101 102 103 104
FITC CD3 -->
10
1
10
2
10
3
10
4
PE
CD
8 --
>
PE
-CD
8
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
PE
CD
8 --
>
PE
-CD
8
TC-CD2
INSTRUMENT COMPENSATION
101 102 103 104
ECD CD4 -->
10
1
10
2
10
3
10
4
PE
CD
8 --
>
PE
-CD
8
ECD-CD4
101 102 103 104
10
1
10
2
10
3
10
4
TC C
D2
-->
TC-C
D2
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
EC
D C
D4
-->
EC
D-C
D4
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
EC
D-C
D4
TC-CD2
PE, TX-RED & PE-CY5COMPENSATION
TC CD2 -->101 102 103 10
10
1
10
2
10
3
10
PE
-CD
8
TC-CD2101 102 103 104
ECD CD4 -->
10
1
10
2
10
3
10
4
PE
-CD
8
ECD-CD4
POOR
GOOD
101 102 103 104
10
1
10
2
10
3
10
4
PE
-CD
8
TC-CD2101 102 103 104
ECD CD4 -->
10
1
10
2
10
3
10
4
PE
-CD
8
ECD-CD4
R3
R3
A C
B D
10 20 30 40 50 60 70 80 90 100 120
FSC -->
1020
3040
5060
7080
90100
120
SS
C -->
10 1 10 2 10 3 10 4
FL1 -->
101
102
103
104
FL2 --> R1
1 2
3 4
FSC
SSC
R1
R2 R3
R4
FITC-CD45
PE
-CD
4
10 1 10 2 10 3 10 4
FL4 -->
101
102
103
104
FL
2 --> R9
9 10
11 12
PE-CY5-CD8P
E-C
D4
R4 R5
R6
10 1 10 2 10 3 10 4
FL1 -->
101
102
103
104
FL3 --> R13
13 14
15 16
PE-CY5-CD8
AP
C-C
D8
R8
R9
R10
COMPENSATION UP TO FOUR COLORS WITH TWO LASERS