Rapid preparation of multiple cell samples for immunofluorescence analysis using microtiter plates

Journal of Immunological Methods, 39 (1980) 85--93

85

Q Elsevier/North-Holland Biomedical Press

RAPID PREPARATION OF MULTIPLE CELL SAMPLES FOR IMMUNOFLUORESCENCE ANALYSIS USING MICROTITER PLATES :

BARBARA UCHAIqSKA-ZIEGLER 1, PETER WERNET and ANDREAS ZIEGLER

Immunology Laboratory, Medical University Clinic, D-7400 Titbingen, F.R.G. (Received 17 April 1980, accepted 18 July 1980)

A method is described which allows incubation, washing and staining of cells for immunofluorescence analysis to be carried out in microtiter plates. Comparison of this procedure with the conventional protocol, carried out on normal and leukemic human peripheral blood cells, reveals 5 major advantages. (1) Only 2.5 × l 0 s viable cells are needed for testing a particular antiserum. (2) The amount of reagents needed is 1/4 of that used in the conventional method. (3) Damage to cells is reduced to a minimum by shorter processing times and gentler centrifugation steps. (4) A large number of samples can be processed at the same time in an identical and reproducible manner. (5) The cost of an experiment is considerably reduced.

INTRODUCTION

Following the introduction of immunofluorescent staining techniques by Coons et al. (1941), a large number of methodological improvements have been devised (Holborow, 1970; Tung, 1977; Knapp, 1978), which have made immunofluorescence an important m e t h o d for the detection and identification of intra- and extracellular antigens (for a recent review see Forni, 1979). Large scale procedures for screening sera b y immunofluorescence with mechanized devices have been reported (O'Neill and Johnson, 1971; Ten Veen et al., 1971), b u t these are unsuitable for small laboratories. Instead, the most frequently used protocol for staining live cells relies on t u b e s for incubation, washing, etc. When many samples have to be processed, this m e t h o d becomes very laborious and time-consuming. In addition, it is difficult to reduce the amounts of cells, antisera and buffers needed to carry out an experiment. In order to overcome these problems, we have used microtiter plates for the preparation of cells for immunofluorescence analysis. The conventional m e t h o d and the new procedure were c o m p a r e d b y reacting periph-

1 In partial fulfillment of Ph.D. thesis requirements. 2 Supported by DFG-Forschergruppe 'Leuk~/mieforschung' Wa 139/11.

86 eral mononuclear cells from healthy and leukemic human donors with several monoclonal antibodies b y indirect immunofluorescence. MATERIAL AND METHODS Cells

Peripheral cells were obtained from 10 healthy donors and from patients with acute myelocytic leukemia (AML), chronic lymphocytic leukemia (CLL) and T-cell acute lymphocytic leukemia (T-ALL). Leukemic cells were selected from the frozen-cell bank of our laboratory. Mononuclear cells were obtained using Ficoll/Hypaque centrifugation (BSyum, 1968). The viability of cells for the immunofluorescent staining procedure was always 98--100%. M o n o c l o n a l an tibodies

W6/32.HLK is a monoclonal mouse IgG antibody, probably of the ~/2a class. It reacts with the heavy chain of HLA-A, B, C antigens when these are associated with ~2-microglobulin (Barnstable et al., 1978). We have used the reactive and non-reactive variants W6/32.HL and W6/32.HK obtained after subcloning of W6/32.HLK which differ only in their light chains (unpublished results, see Ziegler and Milstein, 1979). YD1/63.4.10 and YD1/49.6.8 are doubly cloned m y e l o m a hybrids derived from the fusion of the rat m y e l o m a Y3/Ag1.2.3 (Galfr8 et al., 1979) and spleen cells from a (DA X Lou)Fl rat hyperimmunized with human DAUDI cells (Ziegler et al., manuscript in preparation). Both hybrids secrete IgG; the YD1/63.4.10 antibody appears to react with a c o m m o n determinant on HLA-DR molecules while YD1/49.6.8, used as negative control, does n o t recognize any antigenic determinant present on peripheral mononuclear cells or any leukemic cells tested so far. All m y e l o m a hybrids were grown in Dulbecco's modification of Eagle's Medium supplemented with 10% fetal calf serum (FCS), penicillin (200 E/ml) and streptomycin (200 pg/ml). Tissue culture supernatants were prepared from densely.growing cultures and kept at 4°C after the addition of HEPES buffer to 20 mM and sodium azide to 0.1%. Before immunofluorescence tests, all monoclonal supernatants were centrifuged for 1 h at 144,000 X g. Fluorescein-labeled antisera

For all preparations fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse Ig and an FITC-rabbit anti-rat Ig conjugate (R. Paesel KG, Frankfurt, F.R.G.) were used. To avoid false positive reactions, 200 gl of each conjugated antiserum were mixed with 100 pl of pooled normal human serum for 30 min at RT. After removing precipitates by spinning for 20 min at 1000 X g, the supernatant was diluted 1 : 20 with phosphate-buffered saline,

87 pH 7.3 (PBS), centrifuged for 1 h at 144,000 X g and aliquots stored at - 2 0 ° C in the dark.

Normal rabbit serum (NRS) Pooled serum from unimmunized rabbits was inactivated and then absorbed with pooled cells from AML and CLL patients. 1 ml NRS was mixed with 10 s cells for 30 min at 4°C, spun 10 min at 100 Xg and the supernatant absorbed once again. After an additional low-speed centrifugation, the supernatant was centrifuged for 1 h at 144,000 X g to remove aggregates. The NRS was then stored in aliquots at --20 ° C until use.

Microtiter plate procedure equipment U - b o t t o m flexible polyvinyl chloride microtiter plates (Cat. No. M24), a multichannel reagent dispenser (Cat. No. AM58) and a microshaker (Cat. No. AM69) were all from Dynatech Deutschland GmbH, Plochingen, F.R.G.

Indirect immunofluorescence test (see Fig. 1) (a) Initial preparation of cells. Staining of cells for fluorescence microscopy was carried o u t with viable mononuclear cells in Hank's BSS (HBSS, Gibco-Biocult, Scotland, U.K.) containing 0.5% bovine serum albumin (BSA) and 0.1% sodium azide (HBSS/BSA). Fresh blood was diluted 1 : 2 with HBSS, layered over Ficoll and centrifuged for 25 min at 650 X g. Mononuclear cells were collected from the interphase, washed twice with HBSS/BSA and counted. Frozen cells were thawed at 37°C and resuspended in RPMI medium containing 10% FCS (Associated Biomedic Systems, Inc., Buffalo, NY, U.S.A.). They were then washed twice with HBSS/BSA and counted. Both cell preparations were finally spun and pellets usually containing 107 cells were mixed with 0.4 ml NRS and kept for 20 min on ice. Cells were then adjusted to 107/ml with HBSS/BSA. (b) Tube procedure. 100 pl of cell suspension (106 cells) were pipetted into 1 ml E p p e n d o r f vials. 200 pl of HBSS/BSA were added followed b y a centrifugation at 100 X g for 5 min. The supernatant was aspirated and the pellet resuspended by vortexing. 10 pl of monoclonal antibody supernatant w e r e then added and cells incubated at 4°C for 30 min after which they were washed twice with cold HBSS/BSA. The cell pellets were then mixed with 25 pl of NRS for 5 min at 4°C followed by 25 pl of FITC-anti-Ig for 30 min. After 3 washes, the cell pellet was resuspended in 10 pl of PBS/5% FCS/0.1% sodium azide. 4--5 pl of the cell suspension were finally placed on a slide, covered with a coverslip and sealed with nail varnish. (c) Microtiter plate procedure. 100, 50 or 25 pl of the cell suspension (see "Initial preparation of cells") were pipetted into individual wells of a microtiter plate and 100 pl HBSS/BSA added. After centrifugation for 2 min at 100 × g, the plate was flicked and shaken for 5 sec. 10, 5 or 2.5 pl of monoclonal antibody supernatant were added followed by 30 min incubation at 4°C. After t w o more washing cycles, 25, 12.5 or 6 pl NRS were

88 a d d e d to t h e cells. A f t e r 5 min at 4°C, a c o r r e s p o n d i n g a m o u n t o f FITC-antiIg was a d d e d . A f t e r a f u r t h e r 30 rain at 4°C, the cells were finally washed 3 times and 5 - - 1 0 pl PBS/5% F C S / 0 . 1 % s o d i u m azide added. Slides were p r e p a r e d as described for t h e t u b e p r o t o c o l . F o r each reagent, 2 0 0 - - 4 0 0 viable cells were e x a m i n e d using a Leitz O r t h o l u x m i c r o s c o p e e q u i p p e d with a P l o e m o p a k epi-illuminator and a 150 W lamp. RESULTS

(1) C o m p a r i s o n o f I F t e c h n i q u e in tubes and m i c r o t i t e r plates Peripheral m o n o n u c l e a r cells were p r e p a r e d f r o m 10 h e a l t h y d o n o r s . A f t e r t h e initial cell p r e p a r a t i o n (see Fig. 1), 106 cells were p u t either into t u b e s or into wells o f a m i c r o t i t e r plate. T h e I F staining p r o c e d u r e s were carried o u t as d e p i c t e d in Fig. 1. In o r d e r t o avoid undesirable non-specific reactivity due to F c - r e c e p t o r b i n d i n g a n d / o r c o m p l e x f o r m a t i o n , all antisera were u l t r a c e n t r i f u g e d b e f o r e use, in a d d i t i o n , i n c u b a t i o n steps with n o r m a l serum

IF-Procedure (a) Initial preparation of cells Peripheral blood Isolation of mononuclear cells over Ficoll/Hypaque Wash 2× NRS-- 20 min/4°C $ Adjust_to 10~ eells/rnl

(b) Tube pr*o~edure 100 pl/tube $. . . . 10 pl antibody . . . .

. .

. .

. .

Wash 1>(. .

~

25 pl FITC-anti-Ig . . . . . . . . ___ 10~1 .

.

.

-25/1l NRS 1

.

i~-P~ate procedure

-

-

.

.

.

30 min/4°C . . . . . . -Wash 2 × -

25-100 pl/well $ 2.5-10 pl antibody -~

.......

5 min/4°C . . . . . . .

% 6-25 pl NRS 1

.

.

30 min/4°C- -

6-25 pl FITC-anti-Ig I¢

. .

. .

Wash 3× Resuspend . . . . . .

I

- ~-5:10p~

- --~-*Immunofluoreseence analysis ~ " Fig. 1. Flow sheet showing the preparation of cells for immunofluorescence analysis.

89 TABLE 1 C o m p a r i s o n o f r e a c t i v i t y o f s o m e m o n o c l o n a l a n t i b o d i e s w i t h cells f r o m h e a l t h y individuals p r e p a r e d b y t h e t u b e a n d plate p r o c e d u r e . Donor

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

B.Z. A.Z. P.W. B.B. S.K. K.S. B.G. G.P. J.R. G.M.

W6/32.HL

W6/32.HK

YD1/63.4.10

YD1/49.6.8

Tube 106 cells (%)

Plate 106 cells (%)

Tube 106 cells (%)

Plate 105 cells (%)

Tube 106 cells (%)

Plate 106 cells (%)

Tube 106 cells (%)

Plate 106 cells (%)

96.0 a 95.0 a 100 a 100 a 100 a 100a 100 a 100 a 100 a 60.3 b

100 a 98.5 a 100 a 100 a 100 a 100a 100 a 100 a 100 a 100 b

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

18.2 a 18.4 a 15.6 a 18.6 a 8.7 c 13.3a 18.4 a 22.8 b 17.1 a 15.3 b

17.0 a 16.6 a 14.1 a 16.3 b 7.6 b 12.5a 18.1 a 18.8 b 17.8 a 16.7 b

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

N u m b e r s give p e r c e n t a g e s o f l a b e l e d escence : + d o u b t f u l ring f l u o r e s c e n c e d 1+ v e r y w e a k r i n g f l u o r e s c e n c e ~ c 2+ w e a k ring f l u o r e s c e n c e ~ J b • J 3+ s t r o n g r i n g f l u o r e s c e n c e ~ a 4+ v e r y s t r o n g r i n g f l u o r e s c e n c e ~

cells; t h e s e e x h i b i t d i f f e r e n t s t r e n g t h s o f ring fluorin in in in

table table table table

of the species from which the FITC-conjugated anti-Ig had been prepared (in our case, rabbit), were also carried out. For comparison of the t w o procedures we employed 4 monoclonal antibodies from which t w o (W6/32.HL and W6/32.HK) react with FITC-conjugated rabbit anti-mouse Ig, while YD1/63.4.10 and YD1/49.6.8 react with FITC-conjugated rabbit anti-rat Ig. HLA-A, B, C antigens detected by W6/ 32.HL are present on all nucleated peripheral cells (Brown et al., 1979), while W6/32.HK, the inactive variant, does not exhibit reactivity with the target cells. The same is true for YD1/49.6.8. YD1/63.4.10 reacts with cells expressing antigens controlled by the HLA-D region (Ziegler et al., unpublished results). The results of several experiments are presented in Table 1. It was attempted to evaluate n o t only the percentage of reacting mononuclear cells b u t also to compare the strength of the fluorescence pattern in the two different procedures. In controls, no stained cells were observed. In the case of W6/32.HL, cells from the first, second and tenth donor showed lower values in the tube procedure than expected, while in the plate procedure only the second case gave an unexpectedly low value. Cells from d o n o r G.M., prepared in a tube, were not in good condition and therefore difficult to evaluate. The strength of fluorescence in both procedures appeared to be similar.

S.K. B.B. B.G. K.S. G.P. J.R. G.M.

100 100 100 100 100 100 100

(%)

10 6

a a a a a a b

100 a 100 a 100 a ND ND ND ND

5 × 10 s (%)

ND ND ND 100 a 100 a 100 a 100 a

2.5 X 10 s (%)

W 6 / 3 2 . H L ( n u m b e r o f cells)

0 0 0 0 0 0 0

106 (%) 0 0 0 ND ND ND ND

5 × 10 s (%) ND ND ND 0 0 0 0

2.5 X 10 s (%)

W 6 / 3 2 . H K ( n u m b e r o f cells)

7.6 16.3 18.1 12.5 18.8 17.8 16.7

106 (%)

a a b a b

b

b

7.6 a 15.0 c 18.5 a ND ND ND ND

5 X 10 s (%) ND ND ND 13.4 19.1 17.6 16.5 a b a b

2.5 X 10 s (%)

Y D 1 / 6 3 . 4 . 1 0 ( n u m b e r o f cells)

0 0 0 0 0 0 0

106 (%) 0 0 0 ND ND ND ND

5 X 10 s (%)

ND ND ND 0 0 0 0

2.5 X 10 s (%)

Y D 1 / 4 9 . 6 . 8 ( n u m b e r o f cells)

1. A.M. (T-ALL) 2. P.G. (CLL) 3. K.W. (AML)

Donor

100a

100 a

99.0b

98.0

b

100 c

61.9 c

100 a

100a

100 c

0

0

0

0

0

0

Plate 5x l0 s (%)

0

0

0

Plate 2.5x l0 s (%)

Tube 106 (%)

Plate 2.5x l0 s (%)

Tube 106 (%)

Plate 5x l0 s (%)

W 6 / 3 2 . H K (no. o f cells)

W 6 / 3 2 . H L (no. o f cells)

11.3 a

100a

19.0 c

Tube 106 (%)

12.6 a

100a

12.9 c

Plate 5x l0 s (%)

12.1 a

100a

17.8 c

Plate 2.5x l0 s (%)

Y D 1 / 6 3 . 4 . 1 0 (no. o f cells)

0

0

0

Tube 106 (%)

0

0

0

Plate 5x l0 s (%)

0

0

0

Plate 2.5x l0 s (%)

Y D 1 / 4 9 . 6 . 8 (no. o f cells)

TABLE 3 Reactivity by the t w o p r o c e d u r e s o f various l e u k e m i c cells w i t h m o n o c l o n a l a n t i b o d i e s . N u m b e r s give p e r c e n t a g e s o f cells e x h i b i t i n g ring fluorescence; designation o f t h e i n t e n s i t y o f f l u o r e s c e n c e as in Table 1.

1. 2. 3. 4. 5. 6. 7.

Donor

TABLE 2 Reactivity o f m o n o c l o n a l a n t i b o d i e s in the plate p r o c e d u r e w i t h peripheral m o n o n u c l e a r cells f r o m h e a l t h y individuals. N u m b e r s give percentages o f cells e x h i b i t i n g ring f l u o r e s c e n c e ; d e s i g n a t i o n o f t h e i n t e n s i t y o f f l u o r e s c e n c e as in Table 1. ND = n o t d o n e .

¢D

91 YD1/63.4.10 seemed to react usually with a slightly lower number of cells in the plate than in the tubes. In tubes we generally observed more cells difficult to evaluate than in the plate, which may account for this difference. (2) Reduction o f the amount o f cells and reagents A systematic reduction in the a m o u n t of cells and reagents required was then attempted; only results with the plate procedure are depicted in Table 2, since cell losses did not permit reliable evaluation of the stained population when tubes were used instead. In 3 cases, IF experiments using 5 × l 0 s cells/well ('1/2 plate') were compared with 106 cells/well. Cells from 4 further donors were used for comparison of 2.5 × l 0 s cells ('1/4 plate') and 106 cells/well. The amounts of reagents were also reduced; in case of '1/2 plate' we used half of the usual a m o u n t of reagents, and a quarter the a m o u n t in the '1/4 plate'. In all cases results were virtually identical. Losses of cells following the initial reduction of their n u m b e r per well were negligible; it was never difficult to obtain enough cells to allow comfortable reading of the slides. (3) Comparison carried out on leukemic cells A comparison of both protocols was also carried o u t using cells from leukemic patients. Cells from T-ALL, CLL and AML patients were prepared for immunofluorescence analysis in tubes and in the plate with 106 cells/ well, and in the plate also with 2.5 × 10 s cells/well. Results are presented in Table 3. Only for the T-ALL cells was a much lower value obtained in the tube procedure with W6/32.HL as compared with the plate procedure. The reverse was true for YD1/63.4.10 using 106 cells/well. In both cases, staining of cells was very difficult to evaluate because of clumping and weak reactivity. All other results obtained were identical for the tube, plate and '1/4 plate'. DISCUSSION The aim of this study was to simplify the preparation of cells for immunofluorescence analysis. The conventional tube procedure was compared with a modified protocol in which all incubations, washings, etc. were carried o u t in a microtiter plate. For 106 cells/antiserum the results obtained with cells from several healthy donors (Table 1) illustrate the validity of the plate protocol, no reproducible difference being found between the two methods in percentage of labeled cells. Thus, the plate offers the advantage of processing many samples in an identical manner in a shorter time (Table 4) than the tube procedure. This becomes particularly important when it is intended to exploit the processing capacity of a fluorescence-activated cell sorter. Although we and others have tried repeatedly to reduce the number of cells (results n o t shown) this has been difficult in tubes because of increasing

92 TABLE 4 Comparison of tube and '1/4 plate' procedures for the preparation of cells for immunofluorescence analysis. Critical points considered

'Tube' protocol

'1/4 plate' protocol

1. Number of cells required 2. Amount of reagents required (arbitrary units) 3. Number of samples processed at the same time 4. Cell pellet easily visible during experiment? 5. Time for a complete experiment (see Fig. 1) with 30 samples 6. Suitable for processing normal and leukemic cells? 7. Cost of an experiment

106 1 1 not always

0.25 x 106 0.25 up to 96 always

~3.5 h yes high

~2 h 15 rain yes low

cell losses. In c o n t r a s t , a m a j o r advantage o f t h e m i c r o t i t e r plate p r o t o c o l is t h a t as few as 2.5 X l 0 s cells/antiserum can be used (Table 2). This has considerable relevance to cases w h e r e it is n o t possible to o b t a i n cells in unlimited q u a n t i t y , e.g., f r o m p a t i e n t s in remission f r o m l e u k e m i a , f r o m p a t i e n t s w i t h aplastic anemia, f r o m p a t i e n t s a f t e r b o n e m a r r o w t r a n s p l a n t a t i o n , or children. T h e a m o u n t s o f reagents n e e d e d are c o r r e s p o n d i n g l y r e d u c e d . T h e r e is also a saving in cost w h i c h , even in t h e age o f m o n o c l o n a l antibodies, is an i m p o r t a n t c o n s i d e r a t i o n for l a b o r a t o r i e s n o t involved in t h e p r o d u c t i o n o f these reagents or c o n v e n t i o n a l antisera. O u r a t t e m p t to m o n i t o r t h e strength o f ring f l u o r e s c e n c e (+- t o 4+, see T a b l e 1) can be regarded as o n l y t e n t a t i v e , since t h e r e is a considerable subjective e l e m e n t . A f l u o r e s c e n c e - a c t i v a t e d cell s o r t e r w o u l d o f course avoid this. This s t u d y also shows t h a t p a t h o l o g i c a l cell p o p u l a t i o n s r e a c t in t h e m i c r o t i t e r plate in t h e same w a y as in tubes. Evidence f o r this is p r e s e n t e d in T a b l e 3. AML blasts usually r e a c t with Y D 1 / 6 3 . 4 . 1 0 , a l t h o u g h the unexp e c t e d l y low r e a c t i v i t y o f this m o n o c l o n a l a n t i b o d y w i t h certain AML cells has also b e e n observed in o t h e r A M L patients (Heinrichs et al., u n p u b l i s h e d results); its cause is u n k n o w n . Table 4 summarises t h e respective advantages o f the t w o p r o c e d u r e s . We have used b o t h m e t h o d s in parallel f o r s o m e m o n t h s t o classify l e u k e m i c cell t y p e s with b o t h m o n o c l o n a l and c o n v e n t i o n a l reagents in either d i r e c t or i n d i r e c t i m m u n o f l u o r e s c e n c e and have little d o u b t t h a t t h e ' 1 / 4 p l a t e ' proced u r e offers a n u m b e r o f distinct advantages. NOTE ADDED IN PROOF R e c e n t e x p e r i e n c e indicates t h a t it m a y be possible t o r e d u c e t h e a m o u n t o f cells even f u r t h e r using t h e p r o t o c o l described above.

93 REFERENCES Barnstable, C.J., W.F. Bodmer, G. Brown, G. Galfr~, C. Milstein, A.F. Williams and A. Ziegler, 1978, Cell 14, 9. B6yum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 79. Brown, G., P. Biberfeld, B. Christensson and D.Y. Mason, 1979, Eur. J. Immunol. 9 , 2 7 2 . Coons, A.H., H.J. Creech and R.N. Jons, 1941, Proc. Soc. Exp. Biol. Med. 4 7 , 2 0 0 . Forni, L., 1979, in: Methods in Immunology, eds. I. Lefkovits and B. Pernis (Academic Press, New York) p. 151. Galfr6, G., C. Milstein and B. Wright, 1979, Nature 2 7 7 , 1 3 1 . Holborow, E.J. ed., 1970, Standardization in Immunofluorescence (Blackwell, Oxford). Knapp, W., 1978, in: Immunofluorescence and Related Staining Techniques, eds. W. Knapp, K. Holubar and G. Wick (Elsevier/North-Holland Biomedical Press, Amsterdam) p. 45. O'Neill, P. and G.D. Johnson, 1971, Ann. N.Y. Acad. Sci. 1 7 7 , 4 4 6 . Ten Veen, H.J., A.C.J. Kuivenhoven and T.E.W. Feltkamp, 1971, Ann. N.Y. Acad. Sci. 177,459. Tung, K.S.K., 1977, J. Immunol. Methods 18,391. Ziegler, A. and C. Milstein, 1979, Nature 2 7 9 , 2 4 3 .