MULTIPARAMETER
IMMUNOFLUORESCENCE
Carleton C. Stewart, Ph.D. & Sigrid J. Stewart
R ARKLL
P
aboratory
Cancer Institute
L
OSWE
of
Flow
Cytometry
Department of Health, State of New York
Elm and Carlton Streets
Buffalo, New York 14263
phone: 716-648-5480
fax: 716-648-8806
email: stewart@sc3101.med.buffalo.edu
ANTIBODY BINDING TO CELLS
RPCI
LFC
THE LAW OF MASS ACTION
Ab + Ep
Kf
AbEp
Kf =
Ab Ep
AbEp
Kr
Ab Ep
Kr =
AbEp
Kf range is usually ~ 106
Kr range is usually ~ 10-3
6
Ka = Kf/Kr = 10 /10
-3
= 10
9
WAYS ANTIBODIES BIND TO CELLS
Specific:
Fab to epitope
Fc to Fc receptor
binding is high affinity and saturable
Non Specific:
binding is low affinity and not saturable
Specific Activity is the concentration
of bindable antibody to its epitope
divided by the protein concentration.
SA =
[F(ab') 2]
(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 ANTIBODIES
RPCI
LFC
AMOUNT BOUND
SPECIFIC
ANTIBODY
NON-SPECIFIC
ANTIBODY
CONCENTRATION
3 µg
s/n = 2.5
1 µg
s/n = 2.1
0.3 µg
s/n = 2.4
0.1 µg
s/n = 4.1
0.03 µg
s/n = 4.8
0.01 µg
s/n = 4.6
0.001 µg
s/n = 3.2
auto
3
0.003 µg
s/n = 3.5
5
Signal to Noise
TITER
4
3
2
0
1
2
3
4
5
Dilution
6
7
8
isotype
control
number
antibody
1
10
2
10
3
10
4
10
1
10
2
10
103
4
10
101
102
3
10
cytokeratin
1 µg S/N
Ab 278
IC
5.8
.3 µg S/N
Ab 100
IC
3.6
.01 µg S/N
Ab 25.7
IC
2.6
4
10
Signal to Noise
60
TITER
50
40
30
20
10
0
0
1
2
3
Dilution
4
5
6
number
VERIFICATION OF ANTIBODY COMBINATIONS
10 11
10 22
FITC-CD3
10 33
10 44
10 1
10 2
PE-CD4
10 3
10 4
10 1
10 2
TC-CD8
10 3
10 4
10
10
CONTROL BATCH
4
4
CONTROL BATCH
10
10
3
3
R2
2
2
x = 69
y = 94
10
10
R1
10
10
x = 98
1
1
101
102
103
101
104
102
103
104
TC-CD8
FITC-CD3
10
10
PE- CD4
10
10
10
R2
2
10
2
x = 62 fail
y = 96 pass
3
3
R1
PE-CD4
4
10
4
N EW BATCH
10
x = 97 pass
1
1
10
102
103
104
1
10
2
10
3
10
4
TC-CD8
E-CD4
FITC-CD3
D4
101
BLOCKING IS IMPORTANT
INDIRECT IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody:
murine monoclonal
antibody
Fc
Fab
Second Antibody:
fluoresceinated
goat anti-mouse
IgG F(ab') 2
FcR
A
epitope
B
C
D
ISOTYPE CONTROL- myeloma protein
E
F
G
AUTOFLUORESCENCE CONTROL
H
BLOCKING WITH GOAT IgG
goat IgG
add Mab
Fab
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
LOG FLUORESCENCE
EFFECT OF BLOCKING ON ANTIBODY BINDING TO
MONONUCLEAR CELLS
CELL VOLUME
number
TOTAL
A
C
B
D
channel number
variation in gamma 1 myeloma
protein binding to macrophages
50
45
40
35
30
25
20
15
10
5
0
0
17
39
4
23
mopc myeloma protein
13
21
log fluorescence
Second Reagent Quality
F(ab’)2 of anti IgG
anti IgG
cell volume
DEAD CELLS CAN BE A PROBLEM
• They bind antibodies non-specifically
• They masquerade as specific subsets
• They cause data misinterpretation
ANTIBODIES BIND NONSPECIFICALLY TO DEAD CELLS
PE-LAMBDA
ALL CELLS
VIABLE CELLS
A
B
dead
cells
FL-KAPPA
EMA PROCEDURE
1
lysed, washed
cells
+ 5 µg EMA
2
18 cm.
10 min.
WASH, FIX, AND ANALYZE
3
Evaluating Viability with
Ethidium Monoazide
% dead in gate
= 1%
% dead = 5%
4
120
10
SSC
60
50
SSC
70
40
R9
30
30
40
50
60
70
80
90
100
FSC
120
10
20
Forw ard Scatter Height -->
R9
20
10
Side Scatter Height -->
120
Fluorescence Three Height -->
10
FSC
100
1
20
90
R2
10
30
80
80
40
70
2
50
60
10
60
50
90
70
40
100
80
30
10
90
20
Forward Scatter Height -->
3
100
10
FL3-EMA
120
Side Scatter Height -->
R9
R9
R2
10
20
30
40
50
60
70
80
90
100
Forw ard Scatter Height -->
FSC
120
ONE COLOR IMMUNOPHENOTYPING
RPCI
LFC
SINGLE COLOR IMMUNOPHENOTYPING
1. IgG Block MAB
wash
FL-second antibody F(ab')2 wash
2. IgG Block B-MAB wash
3. IgG Block FL-MAB wash
FL-Avidin
wash
CORRELATED (LIST MODE) DATA ACQUISITION
Entry No.
1
2
3
4
5
6
7
8
k
Value
80
100
40
20
90
120
100
110
50
75
110
120
Cell Number
1
1
1
1
2
2
2
2
n
n
n
n
Parameter
FSC
SSC
Green
Red
FSC
SSC
Green
Red
FSC
SSC
Green
Red
Analysis of List Mode Data
A
number of cells
region A
B
C
forward scatter
CD4 fluorescence
region B
CD4 FLUORESCENCE
region C
STRATEGIES IN MULTICOLOR FLOW
CYTOMETRY
RPCI
LFC
CELLULAR ANTIGENS
cytokines
structure
enzymes
Adhesion
Metabolic
Receptors
courtesy of Jim Bender
FLOW CYTOMETRY VS
MICROSCOPIC IMAGING
s o rt in g c e lls
m o le c u lar qu an t it at io n
an t ige n c o e xpre s s io n
rare c e lls de t e c t io n
c e llu lar in t e rac t io n
s pac ial re lat io n s h ips
c o m part m e n t aliz at io n
s ign al t o n o is e 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
IgG Block + PE-MAB
FL- second antibody F(ab')2
wash
wash
2. IgG Block + B-MAB + FL-MAB
3. IgG Block + FL-MAB + PE- MAB
wash
wash
PE-Avidin
wash
COMBINED INDIRECT AND DIRECT
IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody: mMAB
Second Antibody: FGAM
PE-mMAB
NO BLOCK
CD8 + FGAM
21%
6%
49%
PE-CD4
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
CD8 + FGAM
BLOCKED
12%
42%
PE-CD4
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
10
3
color
10
2
CD3-CD4- black
1
CD3+CD4- blue
CD3-CD4+ cyan
10
FL2-CD4
CD4 -->
10
4
TWO COLOR PATTERN
10
1
10
2
CD3 -->
10
FL1-CD3
3
10
4
CD3+CD4+ green
10
2
1
10
CD4 -->
CD4
2
10
10
2
10
3
10
10
4
1
10
2
CD8 -->
CD3 -->
CD8
2
10
CD8 -->
10
3
10
4
CD3
CD8
1
1
10
10
CD4 -->
10
1
10
CD4
10
3
3
10
10
4
4
THREE COLOR PATTERN
10
1
10
2
CD3 -->
CD3
10
3
10
4
10
3
10
4
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
CD4
3
10
10
2
10
3
10
4
10
1
10
2
10
3
10
10 1
4
10 2
CD3
4
CD3
10
3
1
10
2
CD4
CD4 -->
2
10
CD4 -->
CD4
10
10
2
10
1
10
CD8 -->
10
1
10
CD8
3
3
10
10
10 4
10
4
CD3
10 3
CD3 -->
CD3 -->
CD3 -->
4
1
10
CD4 -->
1
10
1
10
10
10
2
10
3
10
2
CD8
CD8 -->
10
3
10
2
10
1
CD56 -->
CD56
10
4
4
4
FOUR COLOR PATTERN
10
1
10
2
CD56 -->
CD56
10
3
10
4
10
1
10
2
CD56 -->
10
CD56
3
10
4
10
1
10
2
CD8 -->
CD8
10
3
10
4
FL1
FL2
FL2
FL1
FL3
FL3
FL2
FL2
FL3
FL1
FL1
FL3
VISUALIZING EACH POPULATION
FL4
FL4
FL1
FL3
FL2
FL3
FL3
FL1
FL2
FL4
4 COLOR
FL2
3 COLOR
2 COLOR
FL1
BIVARIATE DOT PLOTS CAN BE USED TO DISPLAY THE PATTERNS GENERATED
BY MULTIPARAMETER DATA.
UNDERSTANDING BINARY LOGIC IS USEFUL
P1
+
+
+
+
+
+
+
+
P2
+
+
+
+
+
+
+
+
P3
+
+
+
+
+
+
+
+
P4
+
+
+
+
+
+
+
+
P1
P2
P3
P4
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?
COLOR PATTERNS USED TO VIEW
COEXPRESSION
A
b
2
Ab3
Ab1
A b1
ne
eg
.(-)
os.(+)
ne
g .(-)
no
s.(+)
A
b
1
A
b
2
A b2
neg.(-)
neg.(-)
pos.(+)
p os.(+)
A b3
neg.(-)
neg.(-)
neg.(-)
neg.(-)
color
grey
yellow
cyan
green
A b1
neg.(-)
p os.(+)
neg.(-)
p os.(+)
Ab3
A b2
neg.(-)
neg.(-)
pos.(+)
p os.(+)
A b3
p os.(+)
p os.(+)
pos.(+)
p os.(+)
color
rust
blue
violet
red
Boolean Logic Table For Four Color
Immunophenotyping
R1
R2 R3
R4
bo o le an
Ab1 Ab2 Ab3 Ab4
n o t (R1 o r R2 o r R3 o r R4 )
+
R1 an d n o t (R2 o r R3 o r R4 )
+
R2 an d n o t (R1 o r R3 o r R4 )
+
+
R1 an d R2 an d n o t (R3 o r R4 )
+
R3 an d n o t (R1 o r R2 o r R4 )
+
+
R1 an d R3 an d n o t (R2 o r R4 )
+
+
R2 an d R3 an d n o t (R1 o r R4 )
+
+
+
R1 an d R2 an d R3 an d n o t R4
+ R4 an d n o t (R1 o r R2 o r R3 )
+
+ R4 an d R1 an d n o t (R2 o r R3 )
+
+ R4 an d R2 an d n o t (R1 o r R3 )
+
+
+ R4 an d R1 an d R2 an d n o t R3
+
+ R4 an d R3 an d n o t (R1 o r R2 )
+
+
+ R4 an d R1 an d R3 an d n o t R2
+
+
+ R4 an d R2 an d R3 an d n o t R1
+
+
+
+ R4 an d R2 an d R3 an d R1
Ab4 is t h e gat in g an t ibo dy
c o lo r
gre y
y e llo w
c y an
gre e n
ru s t
blu e
v io le t
re d
gre y
y e llo w
c y an
gre e n
ru s t
blu e
v io le t
re d
Types of Antigen Expression
Ab1
Ab1
Ab1
F
Ab2
E
Ab2
Ab2
D
Ab1
C
Ab2
B
Ab2
Ab2
A
Ab1
Ab1
INTERPRETING COEXPRESSION
4
10
C
D
1
9
A2
1
4
10
C
D
1
9
3
10
2
10
CD19 -->
2
10
CD19 -->
1
10
3
4
10
1
10
2
10
3
CD7 -->
CD7
5
10
20
4
C6
10
C
D
1
9
10
2
10
CD7 -->
21
10
4
3
C
D
7
10
10
10
19
3
1
4
B
18
1
10
2
10
3
CD2 -->
CD2
1
10
D
4
2
3
10
2
10
CD19 -->
R1
1
1
10
10
7
8
10
1
10
2
CD2 -->
CD2
10
3
10
3
4
4
10
1
10
2
CD7
-->
CD7
10
3
10
4
THREE OR MORE COLOR
IMMUNOPHENOTYPING
wash
1. IgG Block + MAB
Biotin- second antibody F(ab')2
wash
wash IgG Block + FL- MAB + PE-MAB + TC- Avidin
2. IgG Block + FL-MAB + PE-MAB + B-MAB
TC-Avidin
wash
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
Low
low-intermediate
high
FLUOROCHROME
phycoerythrin, APC
tandem, CY5
fluorescein, PerCP
STAINING SOP
50 µl
washed, and
blocked*
whole blood or
bone marrow
lyse,
centrifuge,
decant,
blot,
and
resuspend
pellet
Add antibody
combination
15 min. on ice
*add 10 µl mIg (10 mg/ml) to 1 ml washed whole blood.
wash,
fix,
and
analyse
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
B
PerCP-CD4
C
SSC
CD25
CD25
A
PE-CY5-CD4
FSC
PE-fluorescence
Effect of Light Exposure on
PE-CY5 Tandem Fluorescence
TC-CD45
TC-CD3
PE-fluorescence
EFFECT OF BATCH VARIATION
3
10
2
FL2-H -->
10
1
CD32
100
90
80
70
60
SSC-H -->
50
40
SSC
30
10
20
10
120
10
4
Fc Receptor Expression on Blood
Leukocytes
10
20
30
40
50
60
70
80
90
100
120
10
FSC-H -->
1
10
2
10
3
10
4
10
4
FL1-H -->
CD16
10
2
10
3
CD64
FL3-H -->
10
4
3
10
2
10
FL1-H -->
1
1
CD16
10
10
3
10
2
FL2-H -->
10
1
10
CD32
10
10
4
4
FSC
10
1
10
2
10
3
CD64
FL3-H -->
neutrophils
1
10
2
10
3
FL1-H -->
4
10
10
4
10
1
10
10
2
10
3
10
1
10
2
10
3
10
4
FL1-H -->
FL1-H -->
10
1
2
10
10
3
10
4
FL3-H -->
basophils
1
10
2
10
3
10
4
monocytes
10
4
10
1
10
2
10
3
10
4
3
2
1
10
FL2-H -->
10
10
10
10
10
1
FL3-H -->
10
2
10
3
10
4
FL1-H -->
NK cells
10
1
10
2
10
3
10
4
FL3-H -->
4
1
T-cells
10
2
10
3
10
CD16
10
3
10
4
10
2
10
FL2-H -->
1
10
2
10
3
10
4
10
1
4
10
2
10
3
10
4
10
3
10
4
FL3-H -->
B-cells
10
2
FL3-H -->
10
CD64
3
10
4
1
10
2
FL1-H -->
10
CD16
3
10
4
10
2
10
FL2-H -->
10
1
FL2-H -->
1
CD32
CD32
10
2
10
10
3
3
10
4
10
4
3
2
1
FL2-H -->
10
FL1-H -->
1
FL1-H -->
10
CD32
1
10
10
4
FL3-H -->
10
4
10
3
10
2
FL2-H -->
10
1
CD32
10
10
2
1
10
10
4
10
10
2
3
10
10
1
2
FL1-H -->
10
10
10
10
1
10
FL2-H -->
2
1
10
FL2-H -->
10
10
3
3
3
10
10
4
10
3
10
2
10
FL2-H -->
1
10
10
10
4
4
FL1-H -->
2
1
10
1
10
10
10
10
FL2-H -->
10
2
10
FL2-H -->
2
10
1
FL2-H -->
10
3
3
3
10
10
4
4
4
eosinophils
2
1
10
10
4
10
FL2-H -->
10
3
10
2
1
10
1
10
10
10
FL2-H -->
2
10
FL2-H -->
2
10
10
1
FL2-H -->
10
10
3
3
3
10
10
4
4
4
All Cells
10
1
10
2
FL3-H -->
CD64