Journal of Immunological Methods, 9 (1975) 81--84
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A METHOD FOR STAINING MULTIPLE CHEMOTAXIS FILTERS
EDWARD J. LEONARD Tumor Antigen Section, Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
(Received 27 February 1975, accepted 8 May 1975)
A device is described which holds chemotaxis filters on glass slides so that the cells on as many as 128 filters can be stained simultaneously. The procedure for using the batch stainer with Wright's stain is outlined.
INTRODUCTION Studies on c h e m o t a c t i c responses o f l e u k o c y t e s are n o w f r e q u e n t l y perf o r m e d with m o d i f i e d B o y d e n c h a m b e r s in w h i c h r e s p o n d i n g cells migrate t h r o u g h 5 p holes in a p o l y c a r b o n a t e filter and spread o u t o n t h e u n d e r s i d e o f t h e filter ( S n y d e r m a n et al., 1 9 7 2 ; B o e t c h e r and L e o n a r d , 1974). In o u r l a b o r a t o r y t h e filters are t h e n r e m o v e d , placed cell side up on cover slips, air dried and stained with Wright's stain. This p r o c e d u r e allows d i f f e r e n t i a t i o n o f l e u k o c y t e s as readily as in a stained s m e a r o f peripheral blood. T o facilitate staining we have devised a simple rig w h i c h allows us t o stain all the filters f r o m an e x p e r i m e n t s i m u l t a n e o u s l y . MATERIALS AND METHODS T h e staining rig was c o n s t r u c t e d e n t i r e l y o f stainless steel, and consists o f a series o f base plates and finger plates (fig. 1). Base and finger plates were cut t o size with a p o w e r shear f r o m 0.060 inch t h i c k stainless steel. A p o w e r shear was also used t o cut t h e finger plates ( 0 . 0 2 0 inch stainless steel shim stock) and slide guides ( 0 . 0 3 3 inch stainless). T h e holes in t h e plates were p u n c h e d out. T h e b o t t o m base plate was f i t t e d with 2 stainless steel screws, 8 / 3 2 X 1 - - 1 / 2 inch. T h e t h r e a d s o f t h e screws were polished so t h a t t h e o t h e r plates c o u l d slip d o w n t h e screw shafts readily w i t h o u t binding. Fingers and slide guides were s p o t w e l d e d as n o t e d in fig. 1. T h e fingers were b e n t d o w n w a r d slightly. T h e y were l o c a t e d so t h a t t h e ends o f t h e fingers w o u l d a p p l y pressure t o t h e p e r i p h e r y o f t h e filters, m o u n t e d o n slides as s h o w n in fig. 1. A t t h e e n d o f a c h e m o t a x i s e x p e r i m e n t , t h e cells on t h e t o p surface o f t h e filters are wiped a w a y w i t h a c o t t o n swab. T h e filters are t h e n r e m o v e d f r o m
82
Fig. 1. Base plate and finger plate of batch staining rig. One slide with four filters is shown in position. Seven more pairs of base and finger plates can be added. the chambers and placed, migrated cell side up, on 25 X 17 m m glass slides as shown in fig. 1. During the m ount i ng of the filters, the slide rests on a marked template so that the filters can be located properly. There is a space o f 1 m m b e t w e e n adjacent filters. After the filters are air dried, up to 4 slides are positioned on the b o t t o m base plate. The b o t t o m plate has 2 vertical posts, so th at after the slides are in position a finger plate is placed on t op of t h e base plate. The ends of t he fingers will then be in cont act with the out er 2 m m o f the filters. Pairs of base" plates and finger plates are slid down the vertical guide posts for a total of 8 pairs, and the plates are then held together b y turning wing nuts dow n on the guide posts. The completed rig holds up to 32 slides with 4 filters per slide. T he fingers keep the slides in position and prevent filters f r o m floating off during staining.
Reagents and equipment required for staining 1. Two rectangular lucite trays, just large enough to a c c o m m o d a t e the staining rig, oriented with slides in a vertical position. The depth of the slide tray is 39 mm. 2. Fisher certified Wright's stain solution, Cat. No. So-W-16 (Fisher Scientific Co., Pittsburgh, Pa.).
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3. pH 6.8, 0.017 M phosphate buffer. This is made as a 10× concentrated stock solution by dissolving 194 g Na2 HPO4 • 7 H 2 0 and 12.8 g KH2 PO4 in a final volume of 1 liter. Thirteen ml volumes of the stock buffer are distributed into 125 ml bottles and autoclaved. The buffer can then be stored at room temperature and diluted 10-fold before use. 4. Methanol stored in a large wide-mouth bottle for rinsing slide trays and staining rig.
Staining procedure One hundred sixty ml of Wright's stain solution are added to tray No. 1. The staining rig is placed in tray No. 1. F o r t y ml of Wright's stain solution are mixed with 120 ml pH 6.8 buffer and added to tray No. 2. After 75 sec, the staining rig is transferred from tray No. 1 to tray No. 2. After 2 min, tray No. 2 is placed in the sink and the staining solution is flooded out with a stream of demineralized water for a period of about 1 min. The staining rig is dismantled, the slides are removed and allowed to dry. Lucite trays and the loosely reassembled staining rig are washed by dipping into a large widem o u t h e d jar of methanol. The staining solution in tray No. 1 is poured back into a storage bottle; it can be used many times. Losses of strain during transfers are replaced by adding fresh stain up to the 160 ml mark on the storage bottle. RESULTS AND DISCUSSION
The batch stainer enables us to stain up to 128 chemqtaxis filters at the same time. The advantages are speed, uniformity of staining and the excellent differentiation of leukocytes characteristic of Wright's stained preparations. There was no significant difference in numbers of migrated mononuclear cells on replicate filters stained individually or by the bacth method. We now use the batch stainer routinely for both mouse and h u m a n cells. The staining rig is simple in design and can be constructed inexpensively in any shop equipped with a power shear and spot welder. We found that the mixture of stain and buffer for the second step of the Wright's stain procedure could not be used after storage. This is probably due to precipitation of an increasing a m o u n t of stain after buffer is added to the alcoholic stain solution (Lillie, 1969). Therefore the Wright's stain solution and buffer were mixed freshly and discarded after use. In view of the importance of the second step, one may ask whether any staining of the cells occurs during the application of the methanol-stain solutions or whether this is simply an alcohol fixation step. Inspection of blood films after the first step only shows that the cells have taken up dye. Furthermore if methanol alone is substituted for the first step and stain is used only in the second step, staining of the cells is inferior. When chemotaxis filters are swabbed and then removed from chemotaxis
84 c h a m b e r s , cellular debris m a y be left o n t h e t o p surface of t h e filter. Since t h e filter is a p p l i e d t o p surface d o w n to glass slides f o r staining, t h e debris will be t r a p p e d a n d stained. W h e n t h e m i g r a t e d cells on t h e b o t t o m surface o f t h e filter are e x a m i n e d m i c r o s c o p i c a l l y , t h e stained debris m a y cause a b l u r r e d c o l o r e d b a c k g r o u n d t h a t is d i s t u r b i n g to s o m e observers. T h e debris can b e virtually e l i m i n a t e d b y t r a n s f e r r i n g t h e c h e m o t a x i s filters a f t e r staining, while t h e slides are still wet, to a n e w l o c a t i o n on t h e slides. T h e debris a d h e r e s t o t h e glass r a t h e r t h a n t o t h e filter and is t h u s left b e h i n d w h e n t h e filter is m o v e d to a n e w p o s i t i o n .
REFERENCES Boetcher, D.A. and E.J. Leonard, 1974, J. Nat. Cancer Inst. 52, 1091. Lillie, R.D., 1969, H.J. Conn's Biological Stains (Williams and Wilkins, Baltimore) 8th ed., p. 352. Snyderman, R., L.C. Altman, M.S. Hausman and S.E. Mergenhagen, 1972, J. Immunol. 108, 857.
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A METHOD FOR STAINING MULTIPLE CHEMOTAXIS FILTERS
EDWARD J. LEONARD Tumor Antigen Section, Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
(Received 27 February 1975, accepted 8 May 1975)
A device is described which holds chemotaxis filters on glass slides so that the cells on as many as 128 filters can be stained simultaneously. The procedure for using the batch stainer with Wright's stain is outlined.
INTRODUCTION Studies on c h e m o t a c t i c responses o f l e u k o c y t e s are n o w f r e q u e n t l y perf o r m e d with m o d i f i e d B o y d e n c h a m b e r s in w h i c h r e s p o n d i n g cells migrate t h r o u g h 5 p holes in a p o l y c a r b o n a t e filter and spread o u t o n t h e u n d e r s i d e o f t h e filter ( S n y d e r m a n et al., 1 9 7 2 ; B o e t c h e r and L e o n a r d , 1974). In o u r l a b o r a t o r y t h e filters are t h e n r e m o v e d , placed cell side up on cover slips, air dried and stained with Wright's stain. This p r o c e d u r e allows d i f f e r e n t i a t i o n o f l e u k o c y t e s as readily as in a stained s m e a r o f peripheral blood. T o facilitate staining we have devised a simple rig w h i c h allows us t o stain all the filters f r o m an e x p e r i m e n t s i m u l t a n e o u s l y . MATERIALS AND METHODS T h e staining rig was c o n s t r u c t e d e n t i r e l y o f stainless steel, and consists o f a series o f base plates and finger plates (fig. 1). Base and finger plates were cut t o size with a p o w e r shear f r o m 0.060 inch t h i c k stainless steel. A p o w e r shear was also used t o cut t h e finger plates ( 0 . 0 2 0 inch stainless steel shim stock) and slide guides ( 0 . 0 3 3 inch stainless). T h e holes in t h e plates were p u n c h e d out. T h e b o t t o m base plate was f i t t e d with 2 stainless steel screws, 8 / 3 2 X 1 - - 1 / 2 inch. T h e t h r e a d s o f t h e screws were polished so t h a t t h e o t h e r plates c o u l d slip d o w n t h e screw shafts readily w i t h o u t binding. Fingers and slide guides were s p o t w e l d e d as n o t e d in fig. 1. T h e fingers were b e n t d o w n w a r d slightly. T h e y were l o c a t e d so t h a t t h e ends o f t h e fingers w o u l d a p p l y pressure t o t h e p e r i p h e r y o f t h e filters, m o u n t e d o n slides as s h o w n in fig. 1. A t t h e e n d o f a c h e m o t a x i s e x p e r i m e n t , t h e cells on t h e t o p surface o f t h e filters are wiped a w a y w i t h a c o t t o n swab. T h e filters are t h e n r e m o v e d f r o m
82
Fig. 1. Base plate and finger plate of batch staining rig. One slide with four filters is shown in position. Seven more pairs of base and finger plates can be added. the chambers and placed, migrated cell side up, on 25 X 17 m m glass slides as shown in fig. 1. During the m ount i ng of the filters, the slide rests on a marked template so that the filters can be located properly. There is a space o f 1 m m b e t w e e n adjacent filters. After the filters are air dried, up to 4 slides are positioned on the b o t t o m base plate. The b o t t o m plate has 2 vertical posts, so th at after the slides are in position a finger plate is placed on t op of t h e base plate. The ends of t he fingers will then be in cont act with the out er 2 m m o f the filters. Pairs of base" plates and finger plates are slid down the vertical guide posts for a total of 8 pairs, and the plates are then held together b y turning wing nuts dow n on the guide posts. The completed rig holds up to 32 slides with 4 filters per slide. T he fingers keep the slides in position and prevent filters f r o m floating off during staining.
Reagents and equipment required for staining 1. Two rectangular lucite trays, just large enough to a c c o m m o d a t e the staining rig, oriented with slides in a vertical position. The depth of the slide tray is 39 mm. 2. Fisher certified Wright's stain solution, Cat. No. So-W-16 (Fisher Scientific Co., Pittsburgh, Pa.).
83
3. pH 6.8, 0.017 M phosphate buffer. This is made as a 10× concentrated stock solution by dissolving 194 g Na2 HPO4 • 7 H 2 0 and 12.8 g KH2 PO4 in a final volume of 1 liter. Thirteen ml volumes of the stock buffer are distributed into 125 ml bottles and autoclaved. The buffer can then be stored at room temperature and diluted 10-fold before use. 4. Methanol stored in a large wide-mouth bottle for rinsing slide trays and staining rig.
Staining procedure One hundred sixty ml of Wright's stain solution are added to tray No. 1. The staining rig is placed in tray No. 1. F o r t y ml of Wright's stain solution are mixed with 120 ml pH 6.8 buffer and added to tray No. 2. After 75 sec, the staining rig is transferred from tray No. 1 to tray No. 2. After 2 min, tray No. 2 is placed in the sink and the staining solution is flooded out with a stream of demineralized water for a period of about 1 min. The staining rig is dismantled, the slides are removed and allowed to dry. Lucite trays and the loosely reassembled staining rig are washed by dipping into a large widem o u t h e d jar of methanol. The staining solution in tray No. 1 is poured back into a storage bottle; it can be used many times. Losses of strain during transfers are replaced by adding fresh stain up to the 160 ml mark on the storage bottle. RESULTS AND DISCUSSION
The batch stainer enables us to stain up to 128 chemqtaxis filters at the same time. The advantages are speed, uniformity of staining and the excellent differentiation of leukocytes characteristic of Wright's stained preparations. There was no significant difference in numbers of migrated mononuclear cells on replicate filters stained individually or by the bacth method. We now use the batch stainer routinely for both mouse and h u m a n cells. The staining rig is simple in design and can be constructed inexpensively in any shop equipped with a power shear and spot welder. We found that the mixture of stain and buffer for the second step of the Wright's stain procedure could not be used after storage. This is probably due to precipitation of an increasing a m o u n t of stain after buffer is added to the alcoholic stain solution (Lillie, 1969). Therefore the Wright's stain solution and buffer were mixed freshly and discarded after use. In view of the importance of the second step, one may ask whether any staining of the cells occurs during the application of the methanol-stain solutions or whether this is simply an alcohol fixation step. Inspection of blood films after the first step only shows that the cells have taken up dye. Furthermore if methanol alone is substituted for the first step and stain is used only in the second step, staining of the cells is inferior. When chemotaxis filters are swabbed and then removed from chemotaxis
84 c h a m b e r s , cellular debris m a y be left o n t h e t o p surface of t h e filter. Since t h e filter is a p p l i e d t o p surface d o w n to glass slides f o r staining, t h e debris will be t r a p p e d a n d stained. W h e n t h e m i g r a t e d cells on t h e b o t t o m surface o f t h e filter are e x a m i n e d m i c r o s c o p i c a l l y , t h e stained debris m a y cause a b l u r r e d c o l o r e d b a c k g r o u n d t h a t is d i s t u r b i n g to s o m e observers. T h e debris can b e virtually e l i m i n a t e d b y t r a n s f e r r i n g t h e c h e m o t a x i s filters a f t e r staining, while t h e slides are still wet, to a n e w l o c a t i o n on t h e slides. T h e debris a d h e r e s t o t h e glass r a t h e r t h a n t o t h e filter and is t h u s left b e h i n d w h e n t h e filter is m o v e d to a n e w p o s i t i o n .
REFERENCES Boetcher, D.A. and E.J. Leonard, 1974, J. Nat. Cancer Inst. 52, 1091. Lillie, R.D., 1969, H.J. Conn's Biological Stains (Williams and Wilkins, Baltimore) 8th ed., p. 352. Snyderman, R., L.C. Altman, M.S. Hausman and S.E. Mergenhagen, 1972, J. Immunol. 108, 857.
