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 decades 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 L (instead
of recommended 50 L) 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 L 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 IOTest3 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

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

CD5

BL1a

IM0468; IM0469*; IM2637*; IM3627*

57

48

CD7

8H8.1

IM0585; IM1429*; IM3613*

34

58

CD7

3A1E-12H7

6603824;6603822

26

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

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

CD19

HD237 (B4 lytic)

6604551

252

CD20

B9E9

IM1455; IM1451; IM3607*; IM2644; IM3634*; IM3629*

130

CD21

BL13

IM0473

15

CD22

SJ10.1H11

IM0779; IM1835*; IM3704*

11

CD22

HD239 (B3)

6604428

136

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

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

CD45RO

UCHL1

IM1307

191

33

CD49d

HP2/1

IM1404

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

CD59

P282E

IM3457

13

CD63

CLBGran/12

IM1165; IM1914*

38

CD64

22

IM1604; IM3601*; IM3606*

CD65

88H7

IM1654

IL-2

IL-4

TNF

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

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

TCR Pan a-b

BMA031

IM1467; IM2661

238

12

TCR Pan g-d

IMMU 510

IM1571; IM1418; 6607122*;IM2662*

18

TCR g-d

IMMU 515

IM1465

33

TCR-Vb1

BL37.2

IM2406; IM2355*

38

15

TCR-Vb2

MPB2D5

IM2407; IM2213*

35

TCR-Vb5.1

IMMU 157

IM2285

26

TCR-Vb5.2

36213

IM1482; IM2286*

33

TCR-Vb7.1

ZOE

IM2287

84

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

TCR-Vb17

E17.5F3.15.13

IM2048

113

12

TCR-Vb20

ELL1.4

IM2295

70

TCR-Vb21.3

IG125

IM2050

49

TCR-Vb22

IMMU 546

IM2051

80

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

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

67

IM0584; IM1834

37

Activated/Fixed/Frozen
PBMC

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.

KC56 (T200)

65

Application to the broadly used PMA-ionomycin-activated PBMC:

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

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.