Simplified and More Accurate Surface + Intracellular Staining Experiments
by Flow Cytometry.
(And application to frozen activated PBMC)
Fabrice Malergue, Christine Castelli, Michel Herbert,
Cellular Analysis, Beckman Coulter, Inc.
Results
Introduction
Intracellular staining of cells with antibodies requires a fixation step followed by
permeabilization that are known to be deleterious for surface epitopes. This becomes a
problem when the discrimination of cell subsets by surface marker expression is
necessary. Most procedures therefore recommend that the surface staining be
performed before fixation, leading to protocols that are more labour-intensive and time
consuming. Moreover, when multiple monoclonal antibody (mAb) combinations are
used to analyse the same sample, each test must be prepared separately, thus
increasing the likelihood of variations between tubes introduced during the fixation,
washing, or permeabilization steps, and thus decreasing the accuracy of the results. All
these difficulties have hampered the benefit of flow cytometry for the analysis of “the
intracellular domain.”
The dogma that surface epitopes are destroyed by fixation in most cases is being
challenged by the last decade’s advances in flow cytometry: more powerful lasers, more
sensitive detectors, better mAbs, and brighter fluorochromes. Nowadays, the vast
majority of surface molecules can be detected even after the fixation/permeabilization
steps provided that an appropriate mAb clone and/or fluorochrome are used
(preliminary internal data). As expected, the staining intensity of surface markers on
pre-fixed cells is often reduced compared to the non-fixed cells, however, it remains
acceptable in most cases when surface markers are used to gate the cell subsets for
intracellular antigen analysis.
To address this issue and help the users in simplifying their procedure, most BeckmanCoulter conjugated antibodies have been tested after fixation and permeabilization with
the broadly used IntraPrepTM intracellular staining kit. The reagents have been tested on
normal blood and then classified as either “possible use” (sufficient discrimination
between negative and positive cells) or “not recommended”.
Materials and Methods
Reagents:
Conjugated monoclonal antibodies (mAbs): The reagents tested in this study were obtained from Beckman Coulter,
Inc. (BCI), including all the offered anti-human specificities that are currently conjugated with fluorochromes in BCI
catalog. Some markers, however, were excluded, because of their restricted expression to platelets and erythrocytes
(namely CD41, CD42a, CD42b, CD61, CD62P and CD235a). For a comprehensive list of tested mAbs see Tables 1
and 2. These conjugated mAbs were from the IOTest®, Cyto-Stat® / COULTER CLONE® lines of reagents.
Preparation reagents: IntraPrep Permeabilization Reagent kit (Part Number IM2388 or IM2389). VersaLyse Lysing
Solution (PN IM3648), IOTest 3 Fixative Solution (PN IM3515).
Flow Cytometers: Beckman Coulter Cytomics™ FC 500 with RXP software, BD Biosciences FacsCalibur™ with
CellQuest™ software.
Design of the study:
•The standard procedure for intracellular staining using IntraPrep™ Permeabilization Reagent kit includes a fixation
step (Reagent 1), a wash, a permeabilization step (Reagent 2), directly followed by the addition of intracellularspecific mAbs.
•The standard procedure for “combined membrane and intracytoplasmic staining”, as described in IntraPrep insert,
states that cell surface-specific mAbs must be incubated prior to the fixation step.
• Here we investigate the possibility of incubating the cell surface-specific mAbs after the
fixation/wash/permeabilization steps, i.e. simultaneously with intracellular-specific mAbs.
•We have taken advantage of this inversion in the sequence of steps to scale up the fixation / permeabilization
procedure in order to reduce the workload and increase the reproducibility. We choose to treat 500 µL (instead
of recommended 50 µL) whole blood sample at a time in 50 mL conical tubes.
“Fix\Perm-then-Stain” sample preparation:
1.
2.
3.
4.
5.
6.
Put 0.5 mL whole blood sample in a 50 mL conical tube.
Add 1 mL (2 times the blood volume) IntraPrep Reagent 1.
Vigorously vortex. Incubate 15 minutes, RT.
Add 40 mL PBS. Centrifugate 300 x g, 10 minutes.
Discard supernatant. Briefly vortex to resuspend the pellet.
Add 1 mL (2 times the initial blood volume) IntraPrep Reagent 2.
Do NOT vortex. Let mixing occur for 5 minutes.
7.
In parallel, prepare 10 test tubes by putting the desired volume of
surface AND intracellular specific conjugated mAbs.
8.
Mix manually the permeabilized cell suspension and dispatch 100 µL to each test tube.
9.
Vortex gently. Incubate 15 minutes, RT, in the dark.
10. Add 4 mL PBS. Centrifugate 300 x g, 5 minutes.
11. Discard supernatant. Resuspend the pellet with 0.5 mL or 1 mL of IOTest®3 Fixative Solution at its working
concentration (1X), or use PBS containing 0.5 - 0.8% formaldehyde.
The same commercial vials of mAbs are tested in parallel, on the same fresh normal whole blood sample with the
above method and the standard “Stain-then-Lyse” procedure, where VersaLyse is used as lysing reagent.
Data acquisition is performed within 2 hours of preparation. PMTs are set in order to place isotypic control-stained
leucocytes within the first decade of fluorescence. No compensations are applied when single color reagents are
tested individually.
Figure 1: Examples:
“Stain-then-Lyse”
“Fix\Perm-then-Stain”
Isotypic
Control
FL1
MFI: 19,4
26% of total
MFI: 17,2
26% of total
CD3
equivalent
staining
MFI: 15,9
15% of total
CD4
MFI: 33
15% of total
decreased staining
but clear valley
Number(s)
FITC;
PE; ECD;
RMFI:
RMFI:
Std
Procedure
(Cell Line)
Fix\Permthen-Stain
Clone
The “Fix\Perm-then-Stain” procedure does NOT modify the intracellular staining:
CD1a
BL6
IM1942
CD2
39C1.5
IM0442; IM0443*; IM2634; IM3625*
31
Figure 2: CD79a expression in normal B cells and control cells:
CD2
SFCI3Pt2H9 (T11)
6603863;6603849
40
6
CD3
UCHT1
IM1281; IM1282*; IM2705*; IM2635*; IM2467*; 6607100*
65
55
CD4
13B8.2
IM0448; IM0449*; IM2636; IM2468*
53
11
CD4
SFCI12T4D11 (T4)
6603862;6603850;6604727*;6607101*
27
5
CD5
BL1a
IM0468; IM0469*; IM2637*; IM3627*
57
48
CD7
8H8.1
IM0585; IM1429*; IM3613*
34
58
CD7
3A1E-12H7
6603824;6603822
26
7
CD8
B9.11
IM0451; IM0452; IM2638*; IM2469
115
58
CD8
SFCI21Thy2D3 (T8)
6603861;6603848;6604728*;6607011*;6607102*
70
17
CD9
ALB6
IM1755
CD10
ALB1
IM1915; IM3608*; IM2721; IM3633*
CD10
J5
CD11a
CD11b
CD8-APC
« Stain-then-Lyse »
(No permeabilization)
Gated on:
CD8+ T cells:
No perm. New/Normal protocol
CD19+ B cells:
No perm. New/Normal protocol
« Fix\Permthen-Stain »
(New protocol)
CD79a PE
Other non-B cells:
No perm. New/Normal protocol
« Stain-Fix\PermStain »
(Normal protocol)
CD79a PE
Identical staining
Identical background.
CD79a PE
CD19 FITC
Data Interpretation:
The common leucocyte sub-populations (lymphocytes, monocytes, granulocytes) are
discriminated according to their scatter characteristics. Their percentage of positivity and
fluorescence intensity are analyzed. The standard “Stain-then-Lyse” procedure is
considered as a reference. The specificity of the staining in the “Fix\Perm-then-Stain”
procedure is verified by checking the staining pattern of each sub-population (correct
percentage of negative / positive events).
The main criteria to include the conjugated-mAb in Table 2 (“possible use” with the
“Fix\Perm-then-Stain” procedure) is to provide a visible valley between positive and
negative events. The markers are otherwise listed in Table 1 (“not recommended with the
“Fix\Perm-then-Stain” procedure).
Then, the relative mean fluorescence intensity (RMFI; obtained by dividing the MFI of the
positive fraction by the MFI of the negative fraction or provided by the isotypic control) is
indicated to give an objective measurement of the impact of the procedure on the
staining.
Table 1: mAbs classified as “not recommended” with the “Fix\Perm-then-Stain”
procedure
Specificity
Clone
CD8beta
2ST8.5H7
CD16b
1D3
CD23
9P25
CD25
B1.49.9 & 33B3.1 & 1T44H3
CD48
J4.57
CD49b
Gi9
CD54
84H10
CD57
NC1
CD62L
DREG56 & TQ1
CD79b
CB3-1
CD85k
ZM3.8
CD90
F15-42-1-5
CD95
UB2 & 7C11
CD111
R1.302.12
CD112
R2.477.1
CD122
CF1
CD124
S456C9
CD126
M91
CD138
BB4
CD158a
EB6
CD158b
GL183
CD158e
Z27.3.7
CD158i
FES172
CD160
BY55
CD179a
4G7
CD243
UIC2
NKP46
BAB281
CRTH2
BM16
TCR-Va24
C15
TCR-Vb3
CH92
TCR-Vb11
C21
TCR-Vb13.1
IMMU 222
TCR-Vb16
TAMAYA1.2
TCR-Vd2
IMMU 389
TCR-Vg9
IMMU 360
Note that the adverse effect of
formaldehyde fixation is essentially
epitope-specific (NH2 moieties
affected), and not antigen-specific.
Other mAbs may give positive
results for the specificities listed in
Table 1.
Conversely, be aware that other
mAbs than listed in Table 2 may give
negative results for the
corresponding specificities.
Table 2: clones classified “possible use” with the “Fix\Perm-then-Stain” procedure
The different clones tested are indicated as well as the part number(s) of conjugatedmAbs, that are color-coded according to the fluorochrome (FITC, PE, …). FITCconjugates have been tested first, considering that all other conjugates are brighter. If the
FITC-conjugated form is positive, all other fluorochrome conjugates of the same clone are
listed in the table by extrapolation (* marked). If not positive for FITC (or FITC-conjugate
not available), all other available fluorochrome-conjugates have been tested.
SSC
Part
Specificity
For the rare specificities for which the staining shows a continuum between negative and
positive events the percentage of positive cells is decreased; in some cases like CD25,
the negative impact is such that the specificity is removed from the “possible use” Table
2. For other cases, like CD38 and CD69, we decided to keep them, since they can be
highly over-expressed in specific situations (e.g. in vitro activation). The RMFI is then
replaced by an indication of the percent MFI reduction.
Some other specificities are not expressed in normal blood, thus cell lines were used as
target, they are named nearby the RMFI value.
PC5; PC7;
APC
1 0 4(MOLT4)
29
13
40
60
159
92
6604120
58
13
25.3
IM0860; IM1433*
30
21
Bear1
IM0530; IM2581*; IM3611
10
37
CD11b
94 (Mo1)
6602573
27
34
CD11c
BU15
IM1760
106
32
CD13
SJ1D1
IM0778; IM1427
16
7
CD13
Immu103.44
IM2639
83
65
CD13
366 (MY7)
6602989
197
97
CD14
RMO52
IM0645; IM0650; IM2707*; IM2640; IM2580*
193
73
CD14
322A-1 (MY4)
6604110;6603262*
165
CD15
80H5
IM1423; IM1954*; IM2641*
637
CD16
3G8
IM0814; IM1238*; IM2642*; 6607118*
148
12
CD18
7E4
IM1568; IM1570*
29
30
CD19
J4.119
IM1284; IM1285*; IM2708*; IM2643*; IM2470*; IM3628*
45
15
CD19
89B (B4)
6603859;6603846
25
6
CD19
HD237 (B4 lytic)
6604551
252
8
CD20
B9E9
IM1455; IM1451; IM3607*; IM2644; IM3634*; IM3629*
130
6
CD21
BL13
IM0473
15
6
CD22
SJ10.1H11
IM0779; IM1835*; IM3704*
11
7
CD22
HD239 (B3)
6604428
136
8
CD24
ALB9
IM1428
91
67
CD26
BA5
IM1470
61
21
CD27
1A4CD27
IM2578
82
18
CD28
CD28.2
IM2071
118
18
CD29
K20
IM0791
15
10
CD29
4B4
6604105;6604159
CD30
HRS4
IM2033
CD31
1F11
IM2409
60
21
CD32
2E1
IM1935
232
27
CD33
D3HL60.251
IM1135; IM1179*; IM2647*; IM2471*
16
15
CD33
906 (MY9)
6604121
64
15
CD34
581
IM1870; IM1871*; IM2709*; IM2648*; IM2472*
62(KG1A)
CD35
J3D3
IM1836
14
13
CD36
FA6.152
IM0766
141
83
CD37
BL14
IM0457; IM0458
100
CD38
LS198-4-3
IM2371; IM2651
continuum
CD40
MAB89
IM1936
333
CD43
DFT1
IM3264
16
7
CD44
J.173
IM1219
193
86
CD45
J33
IM0782; IM2078; IM2710; IM2653*; IM3548*; IM2473*
404
210
15
1004
16
406 (NK3.3)
70
11
21
7
CD45RO
UCHL1
IM1307
191
33
CD49d
HP2/1
IM1404
8
CD49e
SAM1
IM1854
13
CD50
HP2/19
IM1601
470
CD51
AMF7
IM1855
CD55
JS11KSC2.3
IM2726
161
60
CD56
N901
IM2073; IM2654
105
19
CD58
AICD58
IM1218; IM1430; IM3702*; IM3701*
14
6
CD59
P282E
IM3457
13
CD63
CLBGran/12
IM1165; IM1914*
38
CD64
22
IM1604; IM3601*; IM3606*
CD65
88H7
IM1654
IL-2
IL-4
TNF α
Lymphocytes
CD3
CD3
CD3
CD3
Example 2:
5 Colors (3 gating, 2 cytokines) application on PBMC activated/fixed/frozen:
CD66b
80H3
IM0531
CD69
TP1.55.3
IM1943; IM2656
CD71
YDJ1.2.2
IM0483; IM2001*
62(K562)
CD80
MAB104
IM1976
12
5
CD81
JS64
IM2579
48
33
CD83
HB15a
IM2218; IM3240*
CD86
HA5.2B7
IM2729
36
69
CD89
A3
IM1614
102
21
CD94
HP-3B1
IM2276
50
21
CD100
BD16
IM2730
62
10
CD101
BB27
IM3658
27
12
CD103
2G5
IM1856
27
17
CD116
SCO6
IM1977
51
30
CD117
104D2D1
IM2732; IM2733
CD127
R34.34
IM1980
CD135
SF1.340
IM2234
CD159a
Z199
IM3291
177
15
CD161
191B8
IM3450
49
23
CD203c
97A6
IM3575
15
63
CD244
C1.7
IM1608
94
8
TCR Pan a-b
BMA031
IM1467; IM2661
238
12
TCR Pan g-d
IMMU 510
IM1571; IM1418; 6607122*;IM2662*
18
8
TCR g-d
IMMU 515
IM1465
33
8
TCR-Vb1
BL37.2
IM2406; IM2355*
38
15
TCR-Vb2
MPB2D5
IM2407; IM2213*
35
7
TCR-Vb5.1
IMMU 157
IM2285
26
7
TCR-Vb5.2
36213
IM1482; IM2286*
33
6
TCR-Vb7.1
ZOE
IM2287
84
8
TCR-Vb8
56C5.2
IM2289
117
13
TCR-Vb12
VER2.32.1
IM2291
136
13
TCR-Vb13.6
JU74.3
IM2293
147
13
TCR-Vb14
CAS1.1.3
IM2047
45
6
TCR-Vb17
E17.5F3.15.13
IM2048
113
12
TCR-Vb20
ELL1.4
IM2295
70
9
TCR-Vb21.3
IG125
IM2050
49
9
TCR-Vb22
IMMU 546
IM2051
80
6
TCR-Vd3
P11.5B
IM2005
267
33
HLA-ABC
B9.12.1
IM1838
231
33
HLA-DR, -DP, -DQ
9-49 (I3)
6603424;6604366
201
98
HLA-DR
B8.12.2
IM0463; IM0464*
HLA-DR
Immu-357
IM1638; IM1639*; IM3636*; IM2659*; IM3635*
All cells
7
7
123
0.0
3
0.0
1.5 39.9
monocytes
100.0 0.0
34.4 24.2
Activated
lymphocytes
7
163
45
14
165
320
12(JEA2)
Interferon γ
67
IM0584; IM1834
37
Activated/Fixed/Frozen
PBMC
5
6604104;6603839
463 (MO7E)
Example 1: Standard 2 colors (1 gating, 1 cytokine) applications:
MFI -80%
ALB11
7 9 4(K562)
Following activation, these cells are fragile and a freezing step strongly impairs their
structure. Nevertheless, fixating these cells immediately after activation allows a perfect
conservation when they are further frozen (wash once, resuspend pellet in 1 volume serum, apply 2
volumes IntraPrep Reagent 1, incubate 15 min, wash, resuspend in freezing medium containing DMSO, freeze).
Then, thawed cells are simply washed once, resuspended in IntraPrep Reagent 2, and
distributed in tubes containing the mAbs mixtures.
8
KC56 (T200)
65
Application to the broadly used PMA-ionomycin-activated PBMC:
7
CD45RA
continuum
As expected, most specificities show a decreased RMFI when analyzed according to this
procedure. Nevertheless, the resolution remains acceptable for a large majority of
conjugates.
We believe that most intracellular applications are designed to investigate accurately the
intracellular compartment, and include surface markers only for gating purposes. In these
conditions the « Fix\Perm-then-Stain » procedure does not impact the results, provided
the user has verified that the gating is still adequate. On the contrary, the opportunity to
batch the sample preparation limits the risk of error and the variability of the handlings
within an assay and between experiments, when numerous combinations of markers are
to be tested.
The intended use of these tables is solely to provide a starting point for the researcher
interested in simplifying some intracellular analyses.
223
CD45
6
(A375N)
Interpretation
364
MFI -50%
55
Non-T
0.0
0.0
77.5 22.5
T8
0.2
1.3
62.9 35.6
T4
1.8
0.4
86.2 11.6
742
89
8
7
22
7
105
41
Conclusion
We show a comprehensive list demonstrating that approximately 75% of surface
epitopes can still be detected after fixation / permeabilization with at least one mAb
conjugate. Researchers should in any case confirm that the eventual decreased
surface staining will not affect the analysis in their particular system (e.g. sample type,
culture conditions, preparation method, pathological state, etc.). Obviously, the
intracellular epitopes (not listed) are not affected because they are treated in the
same conditions as the standard IntraPrep protocol. The list allows a “pre-check” on a
particular marker, to determine if the simplified “Fix\Perm-then-Stain” procedure can be
used. Otherwise, the same list allows a researcher to easily create another gating
strategy in order to use the simplified method.
The overwhelming benefits of this simplified procedure are that it allows batching of
cells during the fixation / washing / permeabilization steps, and pre-mixing of cocktails
of surface + intracellular markers.
Combined, these benefits make experiments easier and more accurate.