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A method for the selection of pig lymphocytes bearing surface immunoglobulin
Journal of Immunological Methods, 7 (1975) 251--254 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A METHOD FOR THE SELECTION OF PIG LYMPHOCYTES BEARING SURFACE IMMUNOGLOBULIN
D.B.A. SYMONS and R.K.G. LOKE A. R.C. Institute of Animal Physiology, Babraham, Cam bridge, U.K.
(Received 21 November 1974, accepted 28 November 1974)
Coupling of IgG anti-immunoglobulin to polystyrene bottles with carbodiimide has been used to bind selectively pig lymphocytes previously treated with anti-immunoglobulin or anti-Ig class-specific fluorescent conjugates. Subsequent recovery of the bound cells produced populations with approximately 80% of cells stained for surface Ig and 90% viability.
INTRODUCTION Several t e c h n i q u e s f o r the selection o f l y m p h o c y t e s a c c o r d i n g to t h e i r s u r f a c e p r o p e r t i e s have b e e n devised in r e c e n t y e a r s (Wigzell a n d A n d e r s s o n , 1 9 7 1 ; E d e l m a n et al., 1 9 7 1 ) . B l y m p h o c y t e s c a r r y i n g relatively large a m o u n t s o f surface i m m u n o g l o b u l i n (sIg) h a v e b e e n p r e f e r e n t i a l l y selected b y a d h e r e n c e t o a n t i g l o b u l i n - c o a t e d i m m u n o a b s o r b a n t s ( T h o m a s and Phillips, 1973). A crucial f a c t o r in a n y m e t h o d is the r e c o v e r y o f s u f f i c i e n t n u m b e r s o f viable cells f o r f u r t h e r studies. We r e p o r t a simple m e t h o d f o r the e n r i c h m e n t o f s u r f a c e Ig-bearing pig l y m p h o c y t e s w i t h s u b s e q u e n t m a i n t e n a n c e o f t h e i r viability in culture. MATERIAL AND METHODS Preparation o f a n t i b o d y - c o a t e d bottles
A w a t e r soluble c a r b o d i i m i d e , 1 - c y c l o h e x y l - 3 - ( 2 - m o r p h o l i n o e t h y l ) - c a r b o d i i m i d e m e t h o - p - t o l u e n e s u l f o n a t e (Aldrich), was used to c o u p l e p r o t e i n s t o sterile p o l y s t y r e n e b o t t l e s (50 X 18 m m , 8 ml Bijou, Sterilin, R i c h m o n d , U.K.) b a s e d u p o n the m e t h o d o f E d e l m a n et al. (1971). IgG, p r e p a r e d b y D E A E - c e l l u l o s e c h r o m a t o g r a p h y f r o m pig anti-sheep I g G s e r u m , a n d carb o d i i m i d e w e r e a d d e d t o each b o t t l e in a 1 : 5 w / w ratio and i m m e d i a t e l y dissolved in 2 ml o f 0.15 M NaC1, p H 6.0: o p t i m u m cell b i n d i n g was a c h i e v e d a t 0.5 m g I g G m l w i t h t h e a n t i b o d y used. T h e b o t t l e s were laid o n t h e i r side, w i t h label indicating t h e u p p e r side, so t h a t t h e l o w e r side wall
252 was immersed in the reacting reagents for 1/2--1 hour. T horough washing with three changes o f PBS removed excess reagents from the bottles.
Lymphocyte preparation and fluorescence L y m p h o c y t e s were prepared from pig blood by a m e t h o d previously described (Binns and Symons, 1974). Cells removed from the Ficoll--Triosil interface were washed three times with Eagle's MEM/HEPES (Biocult) and treated with fluorescein isothiocyanate conjugates of either sheep anti-pig IgM, anti-p or anti 7Fc, 0.5 mg/ml for 15 min. After washing, an aliquot of these cells was c o u n t e d by fluorescence microscopy for surface Ig.
Binding and recovery of cells A pp r o x imately 107 of the conjugate-treated cells, in 2 ml Eagle's MEM or RPMI 1640, were added to each bottle, which was laid on its side for 30 min while the cells sedimented ont o the previously coated area. Cells bound to the wall could be observed with a conventional or inverted microscope. Unbound cells were recovered by decanting and gentle washing 3 times with MEM or PBS. Bound cells could be removed either immediately by moderate agitation or by leaving the cells overnight at r o o m t e m p e r a t u r e in RPMI 1640 (+20% ne w bor n calf serum + antibiotics), when m any would detach spontaneously and the remainder could be removed by brief agitation of the bottle contents with a Whirlimixer (Fisons). These could then be examined directly, using fluorescence microscopy, to determine the percentage enrichment. Viability was assessed by exclusion of T rypan blue and/or fluorescein diacetate hydrolysis. Cells for culture were transferred into RPMI 1640 with 20% ne w bor n calf serum and antibiotics in clean bottles.
RESULTS AND DISCUSSION Table 1 shows the degree of enrichment and viability of cells compared with the starting material. Mean viability of 13 samples after recovery and until day 2 was 89.7% dropping to a bout 55% by day 5. The depleted populations were also examined and showed a reduction in selected cells of between 4 and 10 times. The use o f absorbed antisera, specific for IgM and IgG, acted as controls for each other. Incubation of cells with irrelevant antisera e.g. rabbit-anti-pig serum and sheep-anti-rat albumin, pr oduced no significant binding o f cells. A direct m e t h o d of cell binding, with sheep IgG anti-pig IgG bound to the bottle wall and the addition of unt r e a t e d cells, was also tested. Cell binding was good b u t d e t a c h m e n t of bound cells could only be achieved after violent agitation or by scraping the bottle wall; the subsequent viability of cells
253 TABLE 1 Percentage of lymphocytes with surface Ig before and after enrichment. Total Ig% Start
IgM % Enriched
Start
(>95) (99) (95) --
73.3 86.1 84.9 75.0
4.7 15.4 6.8 15.2
Mean 11.7
79.8
4.7 14.8 8.1 19.2
(85) (98) (85) --
10.5
(>95) (99) (95) --
IgG % Enriched
Start
Enriched
73.3 76.1 73.] 75.0
0.3 2.8 0.0 --
n.c. n.c. n.c. --
1.0
--
75.6
(92) (98) (84) --
Total I g % - determined with anti-IgM FITC conjugate, having anti-p and anti-L chain activity; I g M % - determined with specific anti-p FITC; IgG% determined with anti-?Fc FITC. Numbers in parentheses indicate % viability, n.c. -- not counted, owing to low numbers of cells recovered.
selected by this m e t h o d was m u c h l o w e r (30--40%) t h a n b y the indirect m e t h o d described. A l t h o u g h the bijou b o t t l e s used in these e x p e r i m e n t s were n o t ideal f o r viewing cells b o u n d o n the inner surface, t h e y were c h o s e n specifically t o increase the p r o s p e c t o f s u b s e q u e n t successful culture o f the cells. O n c e a d d e d to the bottle, cells c o u l d be m a n i p u l a t e d and solutions c h a n g e d b y sterile t e c h n i q u e s , with the selected cells r e m a i n i n g inside the bottle. A l t h o u g h the direct b i n d i n g m e t h o d was u n s a t i s f a c t o r y for the enrichm e n t o f sIg cells, it m i g h t be useful as a m e a n s o f depleting cell p o p u l a t i o n s o f sIg cells. T h e easier release o f cells b o u n d b y the indirect m e t h o d is p r e s u m a b l y related t o the e x t r a i m m u n o g l o b u l i n link. Initially we w o n d e r e d w h e t h e r the s p o n t a n e o u s release o f cells was associated with r e d i s t r i b u t i o n o f sIg o n the cell while it r e m a i n e d linked t h r o u g h the Ig--anti-Ig c o m p l e x at the t u b e surface: s u b s e q u e n t p i n o c y t o s i s or e x o c y t o s i s o f the c o m p l e x m i g h t have resulted in cell d e t a c h m e n t , since a large p r o p o r t i o n o f r e c o v e r e d cells had internalised the f l u o r e s c e n t Ig--anti-Ig c o m p l e x after o v e r n i g h t i n c u b a t i o n . H o w e v e r , the presence o f s o d i u m azide (3 X 10 -2 M) t h r o u g h o u t , f r o m the first a d d i t i o n o f c o n j u g a t e d antiglobulin to the cells, to inhibit the redistribut i o n o f aggregated c o m p l e x at the cell surface ( T a y l o r et al., 1 9 7 1 ) n e i t h e r p r e v e n t e d cell binding n o r r e d u c e d the ease o f release o f cells b o u n d to the bottle. T h e b i n d i n g o f cells is n o t t h e r e f o r e e n h a n c e d b y prior r e d i s t r i b u t i o n of t h e Ig--anti-Ig c o m p l e x into a cap and it seems unlikely t h a t cells d e t a c h themselves t h r o u g h r e d i s t r i b u t i o n and p i n o c y t o s i s w h e n b o u n d to a solid phase b y Ig--anti-Ig c o m p l e x e s .
254 REFERENCES Binns, R.M. and D.B.A. Symons, 1974, Res. Vet. Sci. 16, 260. Edelman, G.M., U. Rutishauser and C.F. Millette, 1971, Proc. Natl. Acad. Sci. U.S. 68, 2153. Taylor, R.B., W.P.H. Duffus, M.C. Raff and S. de Petris, 1971, Nature New Biol. 233, 225. Thomas, D.B. and B. Phillips, 1973, Eur. J. Immunol. 3, 740. Wigzell, H. and B. Andersson, 1971, Ann. Rev. Micro. 25, 291.
A METHOD FOR THE SELECTION OF PIG LYMPHOCYTES BEARING SURFACE IMMUNOGLOBULIN
D.B.A. SYMONS and R.K.G. LOKE A. R.C. Institute of Animal Physiology, Babraham, Cam bridge, U.K.
(Received 21 November 1974, accepted 28 November 1974)
Coupling of IgG anti-immunoglobulin to polystyrene bottles with carbodiimide has been used to bind selectively pig lymphocytes previously treated with anti-immunoglobulin or anti-Ig class-specific fluorescent conjugates. Subsequent recovery of the bound cells produced populations with approximately 80% of cells stained for surface Ig and 90% viability.
INTRODUCTION Several t e c h n i q u e s f o r the selection o f l y m p h o c y t e s a c c o r d i n g to t h e i r s u r f a c e p r o p e r t i e s have b e e n devised in r e c e n t y e a r s (Wigzell a n d A n d e r s s o n , 1 9 7 1 ; E d e l m a n et al., 1 9 7 1 ) . B l y m p h o c y t e s c a r r y i n g relatively large a m o u n t s o f surface i m m u n o g l o b u l i n (sIg) h a v e b e e n p r e f e r e n t i a l l y selected b y a d h e r e n c e t o a n t i g l o b u l i n - c o a t e d i m m u n o a b s o r b a n t s ( T h o m a s and Phillips, 1973). A crucial f a c t o r in a n y m e t h o d is the r e c o v e r y o f s u f f i c i e n t n u m b e r s o f viable cells f o r f u r t h e r studies. We r e p o r t a simple m e t h o d f o r the e n r i c h m e n t o f s u r f a c e Ig-bearing pig l y m p h o c y t e s w i t h s u b s e q u e n t m a i n t e n a n c e o f t h e i r viability in culture. MATERIAL AND METHODS Preparation o f a n t i b o d y - c o a t e d bottles
A w a t e r soluble c a r b o d i i m i d e , 1 - c y c l o h e x y l - 3 - ( 2 - m o r p h o l i n o e t h y l ) - c a r b o d i i m i d e m e t h o - p - t o l u e n e s u l f o n a t e (Aldrich), was used to c o u p l e p r o t e i n s t o sterile p o l y s t y r e n e b o t t l e s (50 X 18 m m , 8 ml Bijou, Sterilin, R i c h m o n d , U.K.) b a s e d u p o n the m e t h o d o f E d e l m a n et al. (1971). IgG, p r e p a r e d b y D E A E - c e l l u l o s e c h r o m a t o g r a p h y f r o m pig anti-sheep I g G s e r u m , a n d carb o d i i m i d e w e r e a d d e d t o each b o t t l e in a 1 : 5 w / w ratio and i m m e d i a t e l y dissolved in 2 ml o f 0.15 M NaC1, p H 6.0: o p t i m u m cell b i n d i n g was a c h i e v e d a t 0.5 m g I g G m l w i t h t h e a n t i b o d y used. T h e b o t t l e s were laid o n t h e i r side, w i t h label indicating t h e u p p e r side, so t h a t t h e l o w e r side wall
252 was immersed in the reacting reagents for 1/2--1 hour. T horough washing with three changes o f PBS removed excess reagents from the bottles.
Lymphocyte preparation and fluorescence L y m p h o c y t e s were prepared from pig blood by a m e t h o d previously described (Binns and Symons, 1974). Cells removed from the Ficoll--Triosil interface were washed three times with Eagle's MEM/HEPES (Biocult) and treated with fluorescein isothiocyanate conjugates of either sheep anti-pig IgM, anti-p or anti 7Fc, 0.5 mg/ml for 15 min. After washing, an aliquot of these cells was c o u n t e d by fluorescence microscopy for surface Ig.
Binding and recovery of cells A pp r o x imately 107 of the conjugate-treated cells, in 2 ml Eagle's MEM or RPMI 1640, were added to each bottle, which was laid on its side for 30 min while the cells sedimented ont o the previously coated area. Cells bound to the wall could be observed with a conventional or inverted microscope. Unbound cells were recovered by decanting and gentle washing 3 times with MEM or PBS. Bound cells could be removed either immediately by moderate agitation or by leaving the cells overnight at r o o m t e m p e r a t u r e in RPMI 1640 (+20% ne w bor n calf serum + antibiotics), when m any would detach spontaneously and the remainder could be removed by brief agitation of the bottle contents with a Whirlimixer (Fisons). These could then be examined directly, using fluorescence microscopy, to determine the percentage enrichment. Viability was assessed by exclusion of T rypan blue and/or fluorescein diacetate hydrolysis. Cells for culture were transferred into RPMI 1640 with 20% ne w bor n calf serum and antibiotics in clean bottles.
RESULTS AND DISCUSSION Table 1 shows the degree of enrichment and viability of cells compared with the starting material. Mean viability of 13 samples after recovery and until day 2 was 89.7% dropping to a bout 55% by day 5. The depleted populations were also examined and showed a reduction in selected cells of between 4 and 10 times. The use o f absorbed antisera, specific for IgM and IgG, acted as controls for each other. Incubation of cells with irrelevant antisera e.g. rabbit-anti-pig serum and sheep-anti-rat albumin, pr oduced no significant binding o f cells. A direct m e t h o d of cell binding, with sheep IgG anti-pig IgG bound to the bottle wall and the addition of unt r e a t e d cells, was also tested. Cell binding was good b u t d e t a c h m e n t of bound cells could only be achieved after violent agitation or by scraping the bottle wall; the subsequent viability of cells
253 TABLE 1 Percentage of lymphocytes with surface Ig before and after enrichment. Total Ig% Start
IgM % Enriched
Start
(>95) (99) (95) --
73.3 86.1 84.9 75.0
4.7 15.4 6.8 15.2
Mean 11.7
79.8
4.7 14.8 8.1 19.2
(85) (98) (85) --
10.5
(>95) (99) (95) --
IgG % Enriched
Start
Enriched
73.3 76.1 73.] 75.0
0.3 2.8 0.0 --
n.c. n.c. n.c. --
1.0
--
75.6
(92) (98) (84) --
Total I g % - determined with anti-IgM FITC conjugate, having anti-p and anti-L chain activity; I g M % - determined with specific anti-p FITC; IgG% determined with anti-?Fc FITC. Numbers in parentheses indicate % viability, n.c. -- not counted, owing to low numbers of cells recovered.
selected by this m e t h o d was m u c h l o w e r (30--40%) t h a n b y the indirect m e t h o d described. A l t h o u g h the bijou b o t t l e s used in these e x p e r i m e n t s were n o t ideal f o r viewing cells b o u n d o n the inner surface, t h e y were c h o s e n specifically t o increase the p r o s p e c t o f s u b s e q u e n t successful culture o f the cells. O n c e a d d e d to the bottle, cells c o u l d be m a n i p u l a t e d and solutions c h a n g e d b y sterile t e c h n i q u e s , with the selected cells r e m a i n i n g inside the bottle. A l t h o u g h the direct b i n d i n g m e t h o d was u n s a t i s f a c t o r y for the enrichm e n t o f sIg cells, it m i g h t be useful as a m e a n s o f depleting cell p o p u l a t i o n s o f sIg cells. T h e easier release o f cells b o u n d b y the indirect m e t h o d is p r e s u m a b l y related t o the e x t r a i m m u n o g l o b u l i n link. Initially we w o n d e r e d w h e t h e r the s p o n t a n e o u s release o f cells was associated with r e d i s t r i b u t i o n o f sIg o n the cell while it r e m a i n e d linked t h r o u g h the Ig--anti-Ig c o m p l e x at the t u b e surface: s u b s e q u e n t p i n o c y t o s i s or e x o c y t o s i s o f the c o m p l e x m i g h t have resulted in cell d e t a c h m e n t , since a large p r o p o r t i o n o f r e c o v e r e d cells had internalised the f l u o r e s c e n t Ig--anti-Ig c o m p l e x after o v e r n i g h t i n c u b a t i o n . H o w e v e r , the presence o f s o d i u m azide (3 X 10 -2 M) t h r o u g h o u t , f r o m the first a d d i t i o n o f c o n j u g a t e d antiglobulin to the cells, to inhibit the redistribut i o n o f aggregated c o m p l e x at the cell surface ( T a y l o r et al., 1 9 7 1 ) n e i t h e r p r e v e n t e d cell binding n o r r e d u c e d the ease o f release o f cells b o u n d to the bottle. T h e b i n d i n g o f cells is n o t t h e r e f o r e e n h a n c e d b y prior r e d i s t r i b u t i o n of t h e Ig--anti-Ig c o m p l e x into a cap and it seems unlikely t h a t cells d e t a c h themselves t h r o u g h r e d i s t r i b u t i o n and p i n o c y t o s i s w h e n b o u n d to a solid phase b y Ig--anti-Ig c o m p l e x e s .
254 REFERENCES Binns, R.M. and D.B.A. Symons, 1974, Res. Vet. Sci. 16, 260. Edelman, G.M., U. Rutishauser and C.F. Millette, 1971, Proc. Natl. Acad. Sci. U.S. 68, 2153. Taylor, R.B., W.P.H. Duffus, M.C. Raff and S. de Petris, 1971, Nature New Biol. 233, 225. Thomas, D.B. and B. Phillips, 1973, Eur. J. Immunol. 3, 740. Wigzell, H. and B. Andersson, 1971, Ann. Rev. Micro. 25, 291.