Fixation of cell-bound antibody in the membrane immunofluorescence test

Journal of Immunological Methods 4 (1974) 135-148. © North-Holland Publishing Company

FIXATION OF CELL-BOUND ANTIBODY IN THE MEMB RANE IMMUNOFLUORESCENCE TEST P. BIBERFELD, Gunnel BIBERFELD, Zelma MOLNAR* and Astrid FAGRAEUS Departments of Pathology and Immunology, Karolinska Institute, Medical School, National Bacteriological Laboratory, Stockholm, Sweden

Accepted 8 September 1973

Received 14 August 1973

Fixative treatment to stabilize cell-bound antibody was described and applied in quantitative and qualitative studies of cell membrane antigens by direct and indirect immunofluorescence. Of the several fixatives tested, namely p-formaldehyde (PFA), glutaraldehyde (GA) acetone and osmium tetroxide, PFA appeared to have several advantages compared to the other fixatives. Fixation with PFA under specified conditions did not diminish the fluorescence intensity of cells stained by direct or indirect immunofluorescence and decreased only slightly the reaction of anti-immunoglobulin with cell-bound fixed antibody. Redistribution phenomena as 'capping' and dot-formation were abolished or greatly reduced by PFA fixation of the cell-bound antibody in the indirect membrane fluorescence test. The characteristic hand-mirror configuration, due to uropod formation of stimulated lymphocytes was retained if the cells were PFA-fixed at 37°C. After PFA-fixation of stimulated lymphocytes which had been incubated under conditions favouring redistribution of cell-bound antibody it was evident that 'capping' was confined to the side of the cells which formed the uropod.

1. I N T R O D U C T I O N Surface m e m b r a n e antigens o f cells are being extensively studied by the m e m brane i m m u n o f l u o r e s c e n c e technique o f Moiler (1961) or by o t h e r i m m u n o c y t o chemical techniques, w h i c h make use o f living cells. By the i m m u n o f l u o r e s c e n c e t e c h n i q u e , surface antigens, labelled directly or indirectly w i t h fluorescent a n t i b o d y can be easily d e m o n s t r a t e d separately from intracellular antigens, which are n o t labelled due to the i m p e r m e a b i l i t y o f living cells for a n t i b o d y molecules. R e c e n t l y it has been recognized that surface antigens o f living cells in suspension m a y undergo redistribution due to the binding o f a n t i b o d y (Biberfeld et al., 1971; Taylor et al., 1971 ; de Petris et al., 1972; Kourilsky et al., 1972; Loor et al., 1972; Sundqvist, 1972; Unanue et al., 1972a; Wilson et al., 1972). F u r t h e r m o r e a n t i b o d y , which has reacted w i t h surface antigens, can under physiological conditions o f * On leave from: Dept. of Pathology, The University of Chicago, Chicago, Illinois, U.S.A. 135

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incubation be rapidly eliminated from the surface of the living cells by endocytosis (Biberfeld et al., 1971; Unanue et al., 1972b; Wilson et al., 1972). It has also been shown that some antibody, specifically bound to surface antigens may spontaneously dissociate (Jonsson, 1969; Chang et al., 1971, Wilson et al., 1972). Both endocytosis, redistribution and dissociation may affect quantitatively and qualitatively the results of labelling of cell surface antigens. To try to eliminate these phenomena it appeared reasonable to fix the cells in a permanent way as close as possible to the labelling event. Since many membrane antigens arc#damaged by fixation procedures data in preparation), we have investigated the effect of various fixation conditions on surface bound antibodies as used in direct and indirect immunofluorescent reactions.

2. MATERIALS AND METHODS

2.1. Cell suspensions Unless otherwise specified cells from a human lymphoblastoid cell line, Moore 7002, (obtained from Dr. Moore, Roswell Park Memorial Institute, Buffalo, U.S.A.) were used. Human blood lymphocytes were used in some experhnents after purification by sedimentation in gelatin and passage through nylon-wool as previously described (Biberfeld et al., 1971). Mixed lymphocyte cultures (MLC) were set up with purified lymphocytes from two randomly chosen blood donors in a ratio 1:I. All cells were kept in RPMI 1640 culture medium, (Biocult laboratories, Paisley, Scotland) supplemented with 10% foetal calf serum and antibiotics. 2.2. Antisera and FITC-conjugates The antisera used, their source, specificity and concentrations are documented in table 1. All sera obtained from animals were hyperimmune sera. The preparation of

Table 1 Designation

Source

Specificity against

Dilution used

RAHELA RAHOIG SARIG-FITC HCA* (human cold agglufinin serum) SAHU-IgM-FITC ATG-FITC

Rabbit Rabbit Sheep Human

Human (Hela) cells Horse IgG Rabbit IgG I?

1:32 1: 32 1: 5 1:5

Sheep Horse

Human IgM Human (lymphoid) ceils

1:5 1:5

* Sera with high fiter of cold agglutinins from patients with Mycoplasmapneumoniae infection.

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137

immunoglobulins for immunization and conjugation as well as the procedure of conjugation with fluorescein (FITC) has been described elsewhere (Bergquist and Schilling, 1970). All FITC-conjugates were separated from free fluorescein by passage through Sephadex G-25 columns (Bergquist and Schilling, 1970). 2.3. Fixation of antibody treated cells After incubation with antiserum (indirect technique) or with fluorescein (FITC)conjugated antibody (direct technique) the cells (3 to 4 × 10 6/tube) were washed and resuspended in 2 0 - 3 0 vol of fixative for 15 min at 4°C unless otherwise stated. After additional extensive washing cells treated with antiserum were 'stained' with anti-immunoglobulin FITC (anti Ig--EITC). 2.4. Fixatives Unless otherwise stated p-formaldehyde (PFA), reagent grade, was used as a 4% solution in Millonig buffer (Robertson et al., 1963), and glutaraldehyde (GA), (TAAB Laboratories, Reading, England) and osmium tetroxide were made 3 and 2% respectively in 0.1 M cacodylate buffer.

2.5. Fluorescence microscopy, microfluorometry and microphotography A Leitz Orthoplan microscope equipped with incident illumination was used with an iodide 24 V mercury lamp for standard fluorescence microscopy. Microfluorometric measurements on single cells were done with a Leitz MPV microphotometer equipped with a photomultiplier tube with high voltage supply (Jongsma et al., 1971). The light source for microfluorometry was a high pressure xenon arc (XB 75) and excitation filters BG38, TAL 485, selection filters TK495, K495 and barrier filter K510 were used. Readings were recorded with a digital voltmeter (type Optilab Multilinlog 802) and printed out on an electric printer (ADDO-X). The fluorescence intensity index (FI) was calculated for every experiment as the ratio between the mean recorded fluorescence intensity of 2 0 - 3 0 fixed cells over the mean of non-fixed cells treated and measured under identical experimental conditions. Subjectively the fluorescence intensity was scored from 1+ to 4+. Micrographs were taken on Ansco 500 ASA film.

3. RESULTS

3.1. Fixation of cells after staining for membrane immunofluorescence Table 2 shows the effect of treatment with various fixatives on the fluorescence intensity of cells which had been stained either directly with membrane-reactive

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Table 2 Fluorescence index of cells stained by direct or indirect membrane fluorescence and treated with various fixatives. Experiment *

Type of fixation**

Day of microfluorometry after staining and fixation 1

3

4

I II III

PFA, 4% PFA, 4% PFA, 4%

1.04 1.18 1.0z

1.32

I II III

GA, 3~/~ GA, 3% GA, 3~7~

0.92 0.90 0.84

0.81

I II III

Acetone Acetone Osmium, 1%

1.31 1.20 0.06

2.0

1.09

1.02

0.07

* Expt. I direct membrane staining. Expt. II and III indirect membrane staining. ** Cells were fixed for 15 min at +4°C, washed and kept in PBS at +4°C.

F1TC a n t i b o d y or b y the i n d i r e c t t e c h n i q u e prior to f i x a t i o n . The f l u o r e s c e n c e i n d e x ( F I ) o f t h e cells was in m o s t e x p e r i m e n t s slightly h i g h e r t h a n 1 a f t e r P F A or a c e t o n e t r e a t m e n t b u t a f t e r f i x a t i o n w i t h G A usually s o m e w h a t lower. T h e possibility t h a t t h e f i x a t i o n m a d e the cells a u t o f l u o r e s c e n t a n d t h e r e b y c o n t r i b u t e d to the o b s e r v e d increase in FI was investigated b y f l u o r o m e t r i c m e a s u r e m e n t s o f fixed and n o n f i x e d cells. As can b e seen f r o m table 3 a small rise in b a c k g r o u n d fluorescence was o b s e r v e d for cells t r e a t e d w i t h G A over t h a t o f n o n f i x e d cells, b u t n o significant increase was r e c o r d e d f r o m cells fixed in P F A or a c e t o n e , T r e a t m e n t w i t h o s m i u m t e t r a o x i d e a b o l i s h e d virtually all the f l u o r e s c e n c e o f the stained cells. No c o n s p i c u o u s change was o b s e r v e d in t h e ring-type f l u o r e s c e n t p a t t e r n o f the s t a i n e d cells after f i x a t i o n w i t h P F A , G A or a c e t o n e (figs. 1 t o 4).

Table 3 Effect of fixation on cell autofluorescence. Type of fixation

Ratio of cell fluorescence over cell-free background* Expt. 1 Expt. II

-

1.08 1.13 1.42 1.10

PFA 4% GA 3% Acetone

* Mean of 15 20 measurements for each type of treatment.

1.03 1.06 1.29 1.08

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139

Fig. 2.

Fig. 1.

Figs. 1. and 2. Fluorescence microscopy of cells (Moore ~ 7002) stained by indirect immunofluorescence (RAHELA followed by SARIG-FITC) and subsequently treated with various fixtives (as described in Materials and methods): Fig. 1 shows untreated cells and fig. 2 shows cells fixed with PFA.

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Fig. 3.

Fig. 4.

t"igs. 3 and 4. Fluorescence microscopy of cells (Moore 4_ 7002) stained by indirect immunofluorescence (RAIIELA followed by SARIG-t:ITC) and subsequently treated with various fixatives (as described in Materials and Methods): Figs. 3 and 4 show cells fixed with GA and acetone respectively. Two dead cells show intracellular staining.

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141

Table 4 Indirect immunofluorescence of cell-bound antibody after treatment of the cells with various fixatives. Serum

Fixation procedure

Fluoresence pattern* Ring lntracellular**

Negative

H***

RAIIELA RAHELA RAHOIG RAHELA RAHOIG RAHELA RAHOIG RAHELA RAHOIG

4% PFA 4% PFA 3% GA 3% GA Acetone Acetone 2% OSO4 2% OSO4

85 93 0 90 0 89 0 89 90

0 0 92 0 90 0 0 0 0

1 0.6 0.1 0.4 0.2 0.9 0.3 0.1 0.1

5 6 8 10 10 100 100 11 11

Cells: human lymphoblastoid line (Moore v~ 7002). * In per cent (at least 200 cells counted). ** 5 13% showed very strong intracellular fluorescence suggestiveof cell death. *** FI (fluorescence intensity index).

3,2. Indirect membrane immunofluorescence of antibody fixed to the cell membrane Human lymphoblastoid cells treated with rabbit anti-Hela (human) cell serum (RAHELA) or with rabbit anti-horse lg (RAHOIG) as a control were fixed either in p-formaldehyde (PFA), glutaraldehyde (GA), osmium tetraoxide or acetone and subsequently stained with FITC anti-immunoglobulin. Table 4 summarizes the result of such an experiment with regard to the type and intensity of the fluorescent patterns observed after the various fixation procedures used. After fixation with PFA or GA a distinct ring-type fluorescent pattern was observed on cells treated with RAHELA, similar to that of nonfixed ceils. Cells treated with RAHOIG and fixed with PFA or GA did not show surface fluorescence, but a very weak, diffuse cytoplasmic stain. Dead cells, usually 5 - 1 0 % , showed a strong intracellular fluorescence. Fixation with acetone resulted in a strong ring-type fluorescence pattern with RAHELA-treated cells but also diffuse intracellular staining. Cytoplasmic staining was also present in acetone-fixed cells which had been incubated with rabbit anti-horse lg suggesting, that the cells became permeable to antibody after acetone fixation. In contrast, osmium fixed RAHELA-treated cells showed much weaker membrane fluorescence and no cytoplasmic fluorescence. Cells incubated with rabbit anti-horse Ig and fixed with osmium showed a weak nonspecific ring-type membrane fluorescence. Cells fixed with glutaraldehyde or osmium tended to clump, which made fluorometric measurements of single cells difficult.

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1%

• 4%

~.0 rn

• ~n

•

•

•

AlO%

z LAJ

o~ 0.5. 03

W o LL

1 1 4 2

1

5

18 hours

Fig. 5. Microfluorometry of the FITC anti-immunoglobulin reaction with cells treated with RAHELA and fixed with 1.4 or 10% PFA for various periods of time.

The result o f experiments using various concentrations o f P F A and different fixation times are summarized in fig. 5. P F A concentrations up to 10% and fixation times up to 5 hr had little effect on the reaction o f fluorescein-labeled antii m m u n o g l o b u l i n s w i t h the surface b o u n d antibodies. However, after fixation for about 18 hr a r e d u c t i o n in fluorescence intensity i n d e x was observed. 3.3. Test for permeability or fixed cells to antibody and fluorescein F i x a t i o n with P F A , G A or osmium did n o t render cells permeable to a n t i b o d y molecules since fixed cells showed the same ring-type o f m e m b r a n e fluorescence and a p p r o x i m a t e l y the same ,FI as n o n f i x e d cells after t r e a t m e n t with R A H E L A serum and staining w i t h anti-Ig F I T C (table 5). These findings w i t h PFA, GA or osmium fixation also apply to experiments with A T G - F I T C . However, after acet o n e fixation some intracellular penetration o f A T G - F I T C or S A R I G F I T C (table 5) was observed. Free fluorescein p e n e t r a t e d the cells to varying degree b o t h after P F A , G A and acetone fixation (table 5). Table 5 Effect of treatment with various f'txatives on cell membrane permeability to antibody and free fluorescein. Fixation*

4% PFA 10% PFA Acetone 3% GA 2% 0S04

FI** for cells treated with RAHELA and SARIG-FITC

SARIG-FITC

Free fluorescein***

1.05 1.02 1.84 0.60 0.18

0.07 0.03 0.33 0.10 0.02

0.25 0.49 0.18 0.002

* The cells (Moore ~ 7002) were fixed for 15 min. at 4°C. ** FI = fluorescence intensity index. *** Approximately the same concentration as the fluorescein in the SARIC-FITC conjugate.

Fixation of cell-bound antibody

143 % 100

x

Lul.0 D Z

Zos

• 50

(D iJ? W

g 3 u_

O½

12

24

48

72

96 hours

Fig. 6. Cells (Moore ~ 7002 treated with RAHELA, fixed with PFA, incubated at 37°C in culture medium and stained with SARIG-FITC at the time indicated (o). (o) Viable, nonfixed cells. ( - - - ) Per cent cells with ring-type membrane fluorescence. ( ) Fluorescence intensity plotted as fluorescence index of cells measured by microspectrophotometry.

From the decreased FI it was also apparent that GA and osmium fixation o f the cells diminished their reactivity with RAHELA considerably more than did fixation with PFA or acetone (table 4).

3.4. Stability of surface bound antibody after fixation Storage for several days of cells stained directly or indirectly for membrane fluorescence and fixed with PFA (4%, 15 min) did not appreciably diminish their fluorescence intensity (table 2) and most cells retained a ring-type fluorescence pattern. Likewise storage of RAHELA treated and fixed cells for up to 4 days did not appreciably diminish the reaction with fluoresceinated anti-immunoglobulin and over 95% of the cells retained a ring-type of fluorescence pattern (fig. 6). This indicated that most antibody after fixation remained attached to the cell surface during the test-time period. As expected, non-fixed cells which had reacted with RAHELA showed a progressive reduction in binding of sheep anti-rabbit Ig-FITC. The rate of reduction was rapid during the first hour of incubation and levelled off subsequently (fig. 6), indicating that the originally bound antibody was continuously eliminated from the surface of the viable cells. 3.5. Prevention o f cap and dot formation {redistribution) in the indirect immuno17uorescent test by fixation of cell bound antibody Lymphoblastoid cells or peripheral blood lymphocytes treated at 4°C with human sera containing cold agglutinins (HCA) and subsequently with sheep antihuman I g M - F I T C consistently showed a very marked dotted surface fluorescence pattern (Biberfeld et al., 1973a). However, cells fixed after incubation with HCA serum showed a ring-type of fluorescence pattern upon treatment with anti-IgMFITC (figs. 7,8). Similarly Burkitt cells (Raji), stained indirectly for HL-A antigens with multivalent human antiserum (Uguen) showed a discontinuous type of surface

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Fig. 7.

Fig. 8.

Figs. 7 and 8. Indirect immunofluorescence of cells (Moore v~ 7002) incubated with HCA ( h u m a n cold agglutinin serum). Note the dotted pattern of fluorescence (fig. 7). Cells treated with PFA prior to incubation with anti-lgG FITC have a ring-type fluorescence pattern (fig. 8).

Fixation of cell-bound antibody

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Fig. 9. ' H a n d - m i r r o r ' l y m p h o c y t e s f r o m a m i x e d l y m p h o c y t e c u l t u r e i n c u b a t e d w i t h R A H E L A s e r u m at 3 7 ° C f o r 120 rain a n d f i x e d w i t h PI"A p r i o r to s t a i n i n g w i t h a n t i l g C - F I T C . N o t e t h a t t h e f l u o r e s c e n t c a p s c o i n c i d e w i t h the distal p o r t i o n o f t h e u r o p o d s o f t h e cells.

Table 6 'Capping" on uropods of lympocytes. Expt. Temp °C of no.* incubation with anti serum**

Fixation**

4 37 4 4 4 4 4 4

+ + ---+ -

T e m p . °C a n d t i m e (hr) o f p o s t i n c u b a t i o n in complete medium

37 ° 37' 37 °

37 ° 37 °

1 2.5 18 3 18

+ +

Fixation***

% Cells w i t h ' c a p s on uropods §

--+ + +

1 21 22 25 39 0 33 29

* Equal numbers of lymphocytes from two random donors were mixed and cultured for 2 or 3 days before staining. ** R A H E L A . *** 4 % P F A f o r 15 m i n . a t 4 ° C . § M o r e t h a n 9 0 % o f the u r o p o d s w e r e s t r o n g l y s t a i n e d .

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fluorescence when stained alive but an even, ring-type pattern of fluorescence if fixed prior to treatment with FITC anti immunoglobulin. Examination of lymphocytes from mixed lymphocyte cultures (MLC) fixed with PFA or GA before any washing or staining procedures, showed typical 'hand mirror' configuration or uropod formation in 20-30% of the cells. Approximately the same number of uropod-forming cells were also present in cultures incubated with RAHELA provided the cells were kept at 37°C during handling until fixation (table 6). In constrast most cells (unfixed), which had been incubated at 4°C during staining had rounded up (table 6). The lymphocytes which had been treated with RAHELA usually showed a strong concentration of the antibody at the surface of the uropods (table 6). This was particularly evident if the cells were kept at 37°C in culture medium before staining with FITC anti-immunoglobulin (table 6). Thus after 1 - 2 hr' incubation at 37°C 'hand-mirror' lymphocytes were almost exclusively stained over the uropods (fig. 9).

4. DISCUSSION The aim of this study was to find out whether antibody bound to the surface of cells can be stabilized by fixation and yet be demonstrable and quantified by direct and indirect immunofluorescence and microspectrofluorometry. Of the various fixatives tested, namely p-formaldehyde (PEA), glutaraldehyde (GA), acetone and osmium tetraoxide, PFA appeared the most useful for this purpose. It was evident from microspectrofluorometry that PFA treatment did not greatly influence the fluorescence intensity of native cell-bound, fluorescent antibody. The small increase in FI usually observed after fixation may indicate stabilization on the cell surface of antigen-antibody complexes, which in non-fixed preparations tend to dissociate. Autofluorescence and nonspecific binding of free fluorescein may also contribute to the observed increment of the FI, but the role of these factors can easily be assessed under standardized experimental conditions. PFA fixation of antibody bound to the cell membrane moderately decreased the intensity of the reaction with anti-immunoglobulin as judged by quantitative fluorometry. Similar results were also obtained when the reactivity of anti-immunoglobulin for PFA-fixed antibody was tested by passive haemmagghitination and mixed haemadsorption techniques (unpublished data). Of the other fixatives tested acetone made the cells partly permeable to immunoglobulin whereas GA- and osmium-treated cells remained impermeable to antibody molecules. However, GA or osmium fixation significantly decreased the FI of cells stained either by the direct or indirect techniques of membrane fluorescence and the fixed cells became nonspecifically sticky and tended to clump. All fixatives but osmium tetraoxide made the cells permeable to free fluorescein. This makes it necessary to eliminate most of the free fluorescein from the conjugates, for instance by passage through Sephadex columns - as was done during the

Fixation of cell-bound antibody

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present study. However, it is possible that some non-covalently bound fluorescein can remain in the conjugates and dissociate during storage. From the experiments with control (non-reactive) sera it was clear by microspectrofluorometry and also from immuno-electron microscopic observations (data, in preparation) that no measurable nonspecific binding of immunoglobulin to the surface of cells fixed with PFA did occur. PFA-treated cells can for practical purposes be stored for several days without measurable spontaneous dissociation of the fixed antibody. In contrast non-fixed antibody rapidly disappears from the surface of living cells, probably due to dissociation (Chang et al., 1971) and pinocytosis (Biberfeld et al., 1971, Sundqvist, 1972). Fixation of antibody-sensitized cells prevented membrane redistribution of antigen-antibody complexes, which usually occurs on cells stained by the indirect immunofluorescence technique (Biberfeld et al., 1971; Taylor et al., 1971; Davis, 1972; Kourilsky et al., 1972; Loor et al., 1972; Sundqvist, 1972; Wilson et al., 1972). This is consistent with the idea that the redistribution phenomenon is not a passive process but the expression of biological functions of the cell membrane (Taylor et al., 1971 ; Loor et al., 1972). An interesting feature of some lymphocytes is their ability of uropod-formation as seen by the development of a typical 'hand-mirror' configuration (McFarland et al., 1966). This is probably related to an activation and increased mobility of these lymphocytes (Biberfeld, 1971, Biberfeld et al., 1973b). In the present study fixation of cells in mixed lymphocyte cultures, incubated under conditions which induced redistribution of cell-bound antibody showed that cap formation occurred on the side of the cell which is defined by its ability to form a uropod. This is in agreement with the idea that uropods can concentrate cross-linked components of the lymphocyte membrane, a phenomenon which may be important for expression of immunological functions of these cells (De Petris and Raft, 1972, Rosenthal et al., 1973). In conclusion it appears from this study that fixation with p-formaldehyde may be useful in stabilizing cell-bound antibody for quantitative and qualitative studies of membrane antigens by direct and indirect immunofluorescence.

ACKNOWLEDGEMENTS This work was supported by grant 72:70 and 73:78 from the Swedish Cancer Society. The skilled technical assistance of Marianne Ekman, Margareta Andersson, Peder Nilsson, and Magnus Norman is gratefully acknowledged.

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