Journal oflmmunological Methods, 16 (1977) 385--390 © Elsevier/North-Holland Biomedical Press
385
A SIMPLE MULTIPLE CHAMBER APPARATUS FOR MEASURING CHEMOTAXIS OF POLYMORPHONUCLEAR LEUKOCYTES UTILIZING CENTRIFUGATION OF THE CHAMBERS BEFORE INCUBATION 1
MELVIN J. SWANSON 2 Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut 06032, U.S.A.
(Received 20 January 1977; accepted 15 March 1977)
A simple apparatus for measuring chemotaxis is described which contains thirty chambers per unit. Only very few cells and small volumes of chemotactic factor solution are required per chamber. For satisfactory results the apparatus must be centrifuged briefly prior to incubation. Under these conditions results can be obtained that are equivalent to or, with short incubation times, superior to other commonly used modified Boyden chambers. INTRODUCTION M e a s u r e m e n t o f t h e c h e m o t a c t i c r e s p o n s e o f p o l y m o r p h o n u c l e a r leukoc y t e s using c h e m o t a c t i c f a c t o r gradients in filters is a v e r y useful t e c h n i q u e in spite o f b e i n g l a b o r i o u s and having s o m e variables t h a t are d i f f i c u l t t o control. Several m o d i f i c a t i o n s o f B o y d e n c h a m b e r s h a v e b e e n d e s c r i b e d (Ward et al., 1 9 6 5 ; C o m e l y , 1 9 6 6 ; Keller and Sorkin, 1 9 6 8 ; T e m p e l et al., 1 9 7 0 ; Wilkinson, 1 9 7 4 ) . T h e s e v a r y c o n s i d e r a b l y in s i m p l i c i t y , cost, the a m o u n t o f cells and c h e m o t a c t i c f a c t o r t h a t t h e y r e q u i r e and p e r h a p s in the q u a l i t y o f t h e results. A c h e m o t a x i s a p p a r a t u s is d e s c r i b e d in this r e p o r t w h i c h requires v e r y f e w cells and small v o l u m e s o f c h e m o t a c t i c f a c t o r s o l u t i o n , is relatively easy and i n e x p e n s i v e t o c o n s t r u c t , requires m u c h less t i m e to a s s e m b l e and d i s a s s e m b l e c o m p a r e d t o individual c h a m b e r s and gives s a t i s f a c t o r y results w i t h s h o r t e r i n c u b a t i o n t i m e s t h a n o t h e r c o m m o n l y used c h e m o t a x i s c h a m bers. T h e results using this a p p a r a t u s , h o w e v e r , u n d e r t h e c o n d i t i o n s used in this s t u d y , are s a t i s f a c t o r y o n l y w h e n t h e c h a m b e r s are c e n t r i f u g e d p r i o r t o incubation.
1 This work was supported by P.H.S. grants 1P01-AI-12225 and AI09648 awarded to Dr. Elmer L. Becker. z Present address: Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri 64110, U.S.A.
386 MATERIALS AND METHODS
Construction o f apparatus The c h e m o t a x i s c h a m b e r s were m a d e f r o m 0.95 cm thick Plexiglas plates (13 X 11.5 cm). With t w o plates fastened t o g e t h e r , t h i r t y holes o f 0.635 cm d i a m e t e r were drilled t h r o u g h the t o p plate and 0.635 cm d e e p into the bott o m plate using an end mill (which resulted in flat b o t t o m e d holes). The plates were m a r k e d so t h a t t h e y were always assembled in the same positions in which t h e y were drilled. Silicone r u b b e r 0-rings (0.635 cm I.D., Irving B. M o o r e Corp., N e w i n g t o n , C o n n . ) were glued with D o w - C o r n i n g silicone rubber c e m e n t to the b o t t o m s o f t h e u p p e r plates at each hole. Holes were drilled for f o u r bolts to hold the plates t o g e t h e r into which small plastic bolts were glued. The sets o f plates were balanced with each o t h e r b y grinding the plastic along the edges until t h e y were all o f equal weight. Centrifuge holders for t h e c h a m b e r s were c o n s t r u c t e d f r o m materials readily o b t a i n a b l e f r o m a h a r d w a r e store. Strap iron (1.9 cm wide X 0.3 cm thick) was c u t i n t o f o u r lengths o f 13.5 cm and f o u r lengths o f 14.0 cm. Holes were drilled near the ends o f the 14 cm lengths o f strap iron into which 0.6 X 10.5 cm bolts were inserted. Holes were drilled t h r o u g h the 13.5 cm long pieces o f strap iron for small bolts t o hold each pair t o g e t h e r . Also at the centers o f these pieces holes were drilled to a c c o m m o d a t e 0.95 X 7.1 cm rods. Small holes were also drilled t h r o u g h t h e centers of t h e rods into which c o t t e r keys were inserted. Flat washers were placed on each side o f the c o t t e r keys w h e n t h e a p p a r a t u s was assembled as s h o w n in fig. 1. A f t e r c o n s t r u c t i o n , the holders were balanced with each o t h e r b y grinding d o w n the m e t a l slightly at the ends o f the strap iron. The holders were adjusted vertically b y the positioning o f the nuts above and b e l o w the top bars and h o r i z o n t a l l y b y sliding the bolts in or o u t o f the t o p bars until t h e y were level w h e n hanging in the c e n t r i f u g e with the c h a m b e r s c l a m p e d in as s h o w n in fig. 1. Once the holders were adjusted p r o p e r l y all the nuts were t i g h t e n e d a f t e r which n o f u r t h e r a d j u s t m e n t was r e q u i r e d e x c e p t for c e n t e r i n g the c h a m b e r s w h e n t h e y were c l a m p e d in place.
Chemotaxis assay H u m a n n e u t r o p h i l s were isolated b y c e n t r i f u g a t i o n o f b l o o d t h r o u g h disc o n t i n u o u s H y p a q u e - - F i c o l l gradients as previously described (Swanson and Becker). H u m a n C5a was partially purified b y gel filtration o f Z y m o s a n activated serum as previously described ( S w a n s o n and Becker). The C5a was c o n c e n t r a t e d b y l y o p h i l i z a t i o n and desalting on S e p h a d e x G-75. T h e wells o f the multiple c h a m b e r a p p a r a t u s were filled with approxim a t e l y 0.25 ml o f C5a in Tris b u f f e r e d H a n k ' s s o l u t i o n c o n t a i n i n g 1 m g / m l bovine serum albumin. Millipore filters o f 13 m m d i a m e t e r and 3 m i c r o n p o r e size were placed over the wells. T h e u p p e r plates were p o s i t i o n e d and
387
Fig. 1. Photograph of the multiple chamber chemotaxis apparatus. Top) lower (left) and upper (right) plates. Bottom) assembled apparatus in centrifuge holder (left) and centrifuge holder (right).
fastened in place with plastic nuts which were tightened finger tight using a small socket wrench w i t h o u t a handle. The cells (0.3 ml of I X 106 cells/ml) were added to the uppe r c o m p a r t m e n t s , care being taken n o t to have air bubbles trapped in the O-rings. The chambers were then clamped into the centrifuge holders and centrifuged in a Sorvall RC-3 centrifuge equipped with a t y p e HL-4 horizontal r o t o r at 600 rpm (70g) for 2 rain. Immediately after centrifugation the chambers were incubated at 37 ~ C. F o r purposes of comparison, c o m m o n l y used plastic modified Boyden chambers (Tempel et al., 1970) with the lower c o m p a r t m e n t open in a leg of equal height with the cell c o m p a r t m e n t (Ahlco, Inc., Southington, Conn.), referred to here as open plastic chambers, as well as plastic individual blind well chambers (Neuro Probe Inc., Bethesda, Maryland) were also used. The open plastic chambers held a b o u t 1.5 ml of chem ot act i c factor solution whereas the individual blind well chambers held a b o u t 0.25 ml in the wells. Both of these types of chambers were used with the same teflon plugs for the u p p er c o m p a r t m e n t to which was added a p p r o x i m a t e l y 0.7 ml of 7.5 X 10 s cells/ml. The n u m b e r o f cells added t o each of the cham ber types, therefore, was a p p r o x i m a t e l y 1.0 X 106 cells per square c e n t i m e t e r of exposed filter. The individual blind well chambers could be centrifuged in 50 ml plastic tubes by p u t t i ng three #1 r ubber stoppers below t hem in the tubes. Measurement o f cell migration was p e r f o r m e d either by the leading f r o n t
388 m e t h o d ( Z i g m o n d a n d Hirsch, 1 9 7 3 ) or b y c o u n t i n g t h e n u m b e r o f cells in a single p l a n e ( S w a n s o n a n d B e c k e r ) or b y b o t h m e t h o d s . RESULTS C h e m o t a c t i c f a c t o r d o s e r e s p o n s e curves m e a s u r e d b y t w o d i f f e r e n t m e t h o d s a f t e r 30- a n d 60-rain i n c u b a t i o n s are s h o w n in fig. 2. T h e m u l t i p l e c h a m b e r a p p a r a t u s , w h e n c e n t r i f u g e d , gave c o n s i s t e n t l y s t e e p e r dose r e s p o n s e curves t h a n did t h e o p e n plastic c h a m b e r s a f t e r 3 0 - m i n i n c u b a t i o n s . In s o m e e x p e r i m e n t s t h e results o b t a i n e d w i t h t h e o p e n plastic c h a m b e r s with 30-min incubations were unuseable because of insufficient migration w h e r e a s the c e n t r i f u g e d m u l t i p l e c h a m b e r a p p a r a t u s c o n s i s t e n t l y gave s a t i s f a c t o r y results u n d e r t h e s e c o n d i t i o n s . Both the multiple chamber apparatus, when centrifuged, and the open plastic c h a m b e r s quite c o n s i s t e n t l y gave s a t i s f a c t o r y d o s e r e s p o n s e curves a f t e r 6 0 - m i n i n c u b a t i o n s . In s o m e e x p e r i m e n t s t h e m u l t i p l e c h a m b e r a p p a r a t u s gave slightly s t e e p e r d o s e r e s p o n s e curves w h e r e a s in o t h e r s the
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Fig. 2. Chemotaxis dose response curves obtained using the multiple chamber apparatus when centrifuged (open circles), the open plastic chambers (closed circles), the multiple chamber apparatus when not centrifuged (closed triangles) and individual blind well chambers (open triangles). The chambers were incubated for 30 min (A and B) and for 60 min (C and D). The chemotactic response was measured by the leading front method (A and C) and by the number of cells in a single plane (B and D).
389 open plastic chambers gave slightly better results. Usually, however, the results obtained with these two types of chambers after 60-min incubations were approximately equivalent. Fig. 2 also shows data obtained with the multiple chamber apparatus when it was n o t centrifuged. Such dose response curves were usually essentially flat, therefore unuseable. A few points are also shown in fig. 2 that represent data obtained using individual blind well chambers. Although these chambers frequently gave results that were slightly better (i.e. the backgrounds were lower) than those obtained with the multiple chamber apparatus when n o t centrifuged, nevertheless they were still often unsatisfactory. In other experiments (not shown) it was demonstrated that centrifuging the individual blind well chambers increased the differences between background and the m a x i m u m response. The effect of centrifuging the individual blind well chambers, however, was not as dramatic as that of centrifuging the multiple chamber apparatus. DISCUSSION The multiple chamber apparatus used with centrifugation before incubation offers several advantages over most other chemotaxis chambers that have been used in the past. The apparatus can be made at relatively little expense w i t h o u t sophisticated fabricating equipment, therefore is much more economical to obtain than are commercially made chemotaxis chambers. In situations where large numbers of chambers would be required, such as in clinical screening, the time saved in assembling and disassembling the chambers would be significant. There are also circumstances in which one must conserve either chemotactic factor or cells or both. The multiple chamber apparatus requires very few cells per chamber and very small volumes of chemotactic factor solution. The ability to obtain satisfactory results with shorter incubation times than can be obtained with other chambers w i t h o u t centrifugation would also be useful in some applications. A m e t h o d has been described by Baum et al. (1971) and by Hill et al. (1975) that used a cytocentrifuge to deposit cells onto filters before placing them into chemotaxis chambers. Such a method, however, would be much more difficult and time consuming than centrifuging the chambers directly. Hill et al. (1975) concluded that after centrifuging cells onto filters, extracellular protein was not necessary for o p t i m u m migration. The m e t h o d described in this paper, however, does require added protein. The average m a x i m u m response as determined by the leading front m e t h o d in two experiments in which albumin was not added was 28% of the average m a x i m u m response of cells containing 1 mg/ml bovine serum albumin. In fact, an increase in cell migration was observed when albumin was added to the C5a solution as compared to having albumin only in the cell suspension. The reason for the discrepancy in results is unknown. Centrifuging the chambers before incubation eliminates the time required
390 f o r the cells to settle o n t o the filters, t h e r e f o r e increases the migration. The specific reasons, h o w e v e r , w h y c e n t r i f u g a t i o n o f the c h a m b e r s increases the slopes o f dose response curves or w h y the o p e n c h a m b e r s give steeper dose response curves t h a n do blind well c h a m b e r s w h e n n o t c e n t r i f u g e d are n o t k n o w n . Nevertheless, w h e n c e n t r i f u g e d , the results o b t a i n e d using the multiple c h a m b e r a p p a r a t u s are either as g o o d as or b e t t e r t h a n t h o s e o b t a i n e d using the o p e n plastic c h a m b e r s . ACKNOWLEDGEMENTS T h e a u t h o r gratefully a c k n o w l e d g e s the help of Mr. Gerard Gregoire in c o n s t r u c t i o n o f the a p p a r a t u s and o f Mr. H e n r y Showell and Dr. E l m e r B e c k e r f o r helpful discussions and suggestions. REFERENCES Baum, J., A.G. Mowat and J.A. Kirk, 1971, J. Lab. Clin. Med. 77, 501. Cornely, H.P., 1966, Proc. Soc. Exp. Biol. Med. 122,831. Hill, H.R., N.A. Hogan, T.G. Mitchell and P.G. Quie, 1975, J. Lab. Clin. Med. 86, 703. Keller, H.U. and E. Sorkin, 1968, Experientia 24,641. Tempel, T.R., R. Snyderman, H.V. Jordan and S.E. Mergenhagen, 1970, J. Periodontol. 41, 71. Swanson, M.J. and E.L. Becker, 1976, J. Immunol. Methods 13, 191. Ward, P.A., C.G. Cochrane and H.J. Miiller-Eberhard, 1965, J. Exp. Med. 122, 327. Wilkinson, P.C., 1974, Chemotaxis and Inflammation (Churchill Livingstone, Edinburgh and London) pp. 42--45. Zigmond, S.H. and J.G. Hirsch, 1973, J. Exp. Med. 137,387.
385
A SIMPLE MULTIPLE CHAMBER APPARATUS FOR MEASURING CHEMOTAXIS OF POLYMORPHONUCLEAR LEUKOCYTES UTILIZING CENTRIFUGATION OF THE CHAMBERS BEFORE INCUBATION 1
MELVIN J. SWANSON 2 Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut 06032, U.S.A.
(Received 20 January 1977; accepted 15 March 1977)
A simple apparatus for measuring chemotaxis is described which contains thirty chambers per unit. Only very few cells and small volumes of chemotactic factor solution are required per chamber. For satisfactory results the apparatus must be centrifuged briefly prior to incubation. Under these conditions results can be obtained that are equivalent to or, with short incubation times, superior to other commonly used modified Boyden chambers. INTRODUCTION M e a s u r e m e n t o f t h e c h e m o t a c t i c r e s p o n s e o f p o l y m o r p h o n u c l e a r leukoc y t e s using c h e m o t a c t i c f a c t o r gradients in filters is a v e r y useful t e c h n i q u e in spite o f b e i n g l a b o r i o u s and having s o m e variables t h a t are d i f f i c u l t t o control. Several m o d i f i c a t i o n s o f B o y d e n c h a m b e r s h a v e b e e n d e s c r i b e d (Ward et al., 1 9 6 5 ; C o m e l y , 1 9 6 6 ; Keller and Sorkin, 1 9 6 8 ; T e m p e l et al., 1 9 7 0 ; Wilkinson, 1 9 7 4 ) . T h e s e v a r y c o n s i d e r a b l y in s i m p l i c i t y , cost, the a m o u n t o f cells and c h e m o t a c t i c f a c t o r t h a t t h e y r e q u i r e and p e r h a p s in the q u a l i t y o f t h e results. A c h e m o t a x i s a p p a r a t u s is d e s c r i b e d in this r e p o r t w h i c h requires v e r y f e w cells and small v o l u m e s o f c h e m o t a c t i c f a c t o r s o l u t i o n , is relatively easy and i n e x p e n s i v e t o c o n s t r u c t , requires m u c h less t i m e to a s s e m b l e and d i s a s s e m b l e c o m p a r e d t o individual c h a m b e r s and gives s a t i s f a c t o r y results w i t h s h o r t e r i n c u b a t i o n t i m e s t h a n o t h e r c o m m o n l y used c h e m o t a x i s c h a m bers. T h e results using this a p p a r a t u s , h o w e v e r , u n d e r t h e c o n d i t i o n s used in this s t u d y , are s a t i s f a c t o r y o n l y w h e n t h e c h a m b e r s are c e n t r i f u g e d p r i o r t o incubation.
1 This work was supported by P.H.S. grants 1P01-AI-12225 and AI09648 awarded to Dr. Elmer L. Becker. z Present address: Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri 64110, U.S.A.
386 MATERIALS AND METHODS
Construction o f apparatus The c h e m o t a x i s c h a m b e r s were m a d e f r o m 0.95 cm thick Plexiglas plates (13 X 11.5 cm). With t w o plates fastened t o g e t h e r , t h i r t y holes o f 0.635 cm d i a m e t e r were drilled t h r o u g h the t o p plate and 0.635 cm d e e p into the bott o m plate using an end mill (which resulted in flat b o t t o m e d holes). The plates were m a r k e d so t h a t t h e y were always assembled in the same positions in which t h e y were drilled. Silicone r u b b e r 0-rings (0.635 cm I.D., Irving B. M o o r e Corp., N e w i n g t o n , C o n n . ) were glued with D o w - C o r n i n g silicone rubber c e m e n t to the b o t t o m s o f t h e u p p e r plates at each hole. Holes were drilled for f o u r bolts to hold the plates t o g e t h e r into which small plastic bolts were glued. The sets o f plates were balanced with each o t h e r b y grinding the plastic along the edges until t h e y were all o f equal weight. Centrifuge holders for t h e c h a m b e r s were c o n s t r u c t e d f r o m materials readily o b t a i n a b l e f r o m a h a r d w a r e store. Strap iron (1.9 cm wide X 0.3 cm thick) was c u t i n t o f o u r lengths o f 13.5 cm and f o u r lengths o f 14.0 cm. Holes were drilled near the ends o f the 14 cm lengths o f strap iron into which 0.6 X 10.5 cm bolts were inserted. Holes were drilled t h r o u g h the 13.5 cm long pieces o f strap iron for small bolts t o hold each pair t o g e t h e r . Also at the centers o f these pieces holes were drilled to a c c o m m o d a t e 0.95 X 7.1 cm rods. Small holes were also drilled t h r o u g h t h e centers of t h e rods into which c o t t e r keys were inserted. Flat washers were placed on each side o f the c o t t e r keys w h e n t h e a p p a r a t u s was assembled as s h o w n in fig. 1. A f t e r c o n s t r u c t i o n , the holders were balanced with each o t h e r b y grinding d o w n the m e t a l slightly at the ends o f the strap iron. The holders were adjusted vertically b y the positioning o f the nuts above and b e l o w the top bars and h o r i z o n t a l l y b y sliding the bolts in or o u t o f the t o p bars until t h e y were level w h e n hanging in the c e n t r i f u g e with the c h a m b e r s c l a m p e d in as s h o w n in fig. 1. Once the holders were adjusted p r o p e r l y all the nuts were t i g h t e n e d a f t e r which n o f u r t h e r a d j u s t m e n t was r e q u i r e d e x c e p t for c e n t e r i n g the c h a m b e r s w h e n t h e y were c l a m p e d in place.
Chemotaxis assay H u m a n n e u t r o p h i l s were isolated b y c e n t r i f u g a t i o n o f b l o o d t h r o u g h disc o n t i n u o u s H y p a q u e - - F i c o l l gradients as previously described (Swanson and Becker). H u m a n C5a was partially purified b y gel filtration o f Z y m o s a n activated serum as previously described ( S w a n s o n and Becker). The C5a was c o n c e n t r a t e d b y l y o p h i l i z a t i o n and desalting on S e p h a d e x G-75. T h e wells o f the multiple c h a m b e r a p p a r a t u s were filled with approxim a t e l y 0.25 ml o f C5a in Tris b u f f e r e d H a n k ' s s o l u t i o n c o n t a i n i n g 1 m g / m l bovine serum albumin. Millipore filters o f 13 m m d i a m e t e r and 3 m i c r o n p o r e size were placed over the wells. T h e u p p e r plates were p o s i t i o n e d and
387
Fig. 1. Photograph of the multiple chamber chemotaxis apparatus. Top) lower (left) and upper (right) plates. Bottom) assembled apparatus in centrifuge holder (left) and centrifuge holder (right).
fastened in place with plastic nuts which were tightened finger tight using a small socket wrench w i t h o u t a handle. The cells (0.3 ml of I X 106 cells/ml) were added to the uppe r c o m p a r t m e n t s , care being taken n o t to have air bubbles trapped in the O-rings. The chambers were then clamped into the centrifuge holders and centrifuged in a Sorvall RC-3 centrifuge equipped with a t y p e HL-4 horizontal r o t o r at 600 rpm (70g) for 2 rain. Immediately after centrifugation the chambers were incubated at 37 ~ C. F o r purposes of comparison, c o m m o n l y used plastic modified Boyden chambers (Tempel et al., 1970) with the lower c o m p a r t m e n t open in a leg of equal height with the cell c o m p a r t m e n t (Ahlco, Inc., Southington, Conn.), referred to here as open plastic chambers, as well as plastic individual blind well chambers (Neuro Probe Inc., Bethesda, Maryland) were also used. The open plastic chambers held a b o u t 1.5 ml of chem ot act i c factor solution whereas the individual blind well chambers held a b o u t 0.25 ml in the wells. Both of these types of chambers were used with the same teflon plugs for the u p p er c o m p a r t m e n t to which was added a p p r o x i m a t e l y 0.7 ml of 7.5 X 10 s cells/ml. The n u m b e r o f cells added t o each of the cham ber types, therefore, was a p p r o x i m a t e l y 1.0 X 106 cells per square c e n t i m e t e r of exposed filter. The individual blind well chambers could be centrifuged in 50 ml plastic tubes by p u t t i ng three #1 r ubber stoppers below t hem in the tubes. Measurement o f cell migration was p e r f o r m e d either by the leading f r o n t
388 m e t h o d ( Z i g m o n d a n d Hirsch, 1 9 7 3 ) or b y c o u n t i n g t h e n u m b e r o f cells in a single p l a n e ( S w a n s o n a n d B e c k e r ) or b y b o t h m e t h o d s . RESULTS C h e m o t a c t i c f a c t o r d o s e r e s p o n s e curves m e a s u r e d b y t w o d i f f e r e n t m e t h o d s a f t e r 30- a n d 60-rain i n c u b a t i o n s are s h o w n in fig. 2. T h e m u l t i p l e c h a m b e r a p p a r a t u s , w h e n c e n t r i f u g e d , gave c o n s i s t e n t l y s t e e p e r dose r e s p o n s e curves t h a n did t h e o p e n plastic c h a m b e r s a f t e r 3 0 - m i n i n c u b a t i o n s . In s o m e e x p e r i m e n t s t h e results o b t a i n e d w i t h t h e o p e n plastic c h a m b e r s with 30-min incubations were unuseable because of insufficient migration w h e r e a s the c e n t r i f u g e d m u l t i p l e c h a m b e r a p p a r a t u s c o n s i s t e n t l y gave s a t i s f a c t o r y results u n d e r t h e s e c o n d i t i o n s . Both the multiple chamber apparatus, when centrifuged, and the open plastic c h a m b e r s quite c o n s i s t e n t l y gave s a t i s f a c t o r y d o s e r e s p o n s e curves a f t e r 6 0 - m i n i n c u b a t i o n s . In s o m e e x p e r i m e n t s t h e m u l t i p l e c h a m b e r a p p a r a t u s gave slightly s t e e p e r d o s e r e s p o n s e curves w h e r e a s in o t h e r s the
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Fig. 2. Chemotaxis dose response curves obtained using the multiple chamber apparatus when centrifuged (open circles), the open plastic chambers (closed circles), the multiple chamber apparatus when not centrifuged (closed triangles) and individual blind well chambers (open triangles). The chambers were incubated for 30 min (A and B) and for 60 min (C and D). The chemotactic response was measured by the leading front method (A and C) and by the number of cells in a single plane (B and D).
389 open plastic chambers gave slightly better results. Usually, however, the results obtained with these two types of chambers after 60-min incubations were approximately equivalent. Fig. 2 also shows data obtained with the multiple chamber apparatus when it was n o t centrifuged. Such dose response curves were usually essentially flat, therefore unuseable. A few points are also shown in fig. 2 that represent data obtained using individual blind well chambers. Although these chambers frequently gave results that were slightly better (i.e. the backgrounds were lower) than those obtained with the multiple chamber apparatus when n o t centrifuged, nevertheless they were still often unsatisfactory. In other experiments (not shown) it was demonstrated that centrifuging the individual blind well chambers increased the differences between background and the m a x i m u m response. The effect of centrifuging the individual blind well chambers, however, was not as dramatic as that of centrifuging the multiple chamber apparatus. DISCUSSION The multiple chamber apparatus used with centrifugation before incubation offers several advantages over most other chemotaxis chambers that have been used in the past. The apparatus can be made at relatively little expense w i t h o u t sophisticated fabricating equipment, therefore is much more economical to obtain than are commercially made chemotaxis chambers. In situations where large numbers of chambers would be required, such as in clinical screening, the time saved in assembling and disassembling the chambers would be significant. There are also circumstances in which one must conserve either chemotactic factor or cells or both. The multiple chamber apparatus requires very few cells per chamber and very small volumes of chemotactic factor solution. The ability to obtain satisfactory results with shorter incubation times than can be obtained with other chambers w i t h o u t centrifugation would also be useful in some applications. A m e t h o d has been described by Baum et al. (1971) and by Hill et al. (1975) that used a cytocentrifuge to deposit cells onto filters before placing them into chemotaxis chambers. Such a method, however, would be much more difficult and time consuming than centrifuging the chambers directly. Hill et al. (1975) concluded that after centrifuging cells onto filters, extracellular protein was not necessary for o p t i m u m migration. The m e t h o d described in this paper, however, does require added protein. The average m a x i m u m response as determined by the leading front m e t h o d in two experiments in which albumin was not added was 28% of the average m a x i m u m response of cells containing 1 mg/ml bovine serum albumin. In fact, an increase in cell migration was observed when albumin was added to the C5a solution as compared to having albumin only in the cell suspension. The reason for the discrepancy in results is unknown. Centrifuging the chambers before incubation eliminates the time required
390 f o r the cells to settle o n t o the filters, t h e r e f o r e increases the migration. The specific reasons, h o w e v e r , w h y c e n t r i f u g a t i o n o f the c h a m b e r s increases the slopes o f dose response curves or w h y the o p e n c h a m b e r s give steeper dose response curves t h a n do blind well c h a m b e r s w h e n n o t c e n t r i f u g e d are n o t k n o w n . Nevertheless, w h e n c e n t r i f u g e d , the results o b t a i n e d using the multiple c h a m b e r a p p a r a t u s are either as g o o d as or b e t t e r t h a n t h o s e o b t a i n e d using the o p e n plastic c h a m b e r s . ACKNOWLEDGEMENTS T h e a u t h o r gratefully a c k n o w l e d g e s the help of Mr. Gerard Gregoire in c o n s t r u c t i o n o f the a p p a r a t u s and o f Mr. H e n r y Showell and Dr. E l m e r B e c k e r f o r helpful discussions and suggestions. REFERENCES Baum, J., A.G. Mowat and J.A. Kirk, 1971, J. Lab. Clin. Med. 77, 501. Cornely, H.P., 1966, Proc. Soc. Exp. Biol. Med. 122,831. Hill, H.R., N.A. Hogan, T.G. Mitchell and P.G. Quie, 1975, J. Lab. Clin. Med. 86, 703. Keller, H.U. and E. Sorkin, 1968, Experientia 24,641. Tempel, T.R., R. Snyderman, H.V. Jordan and S.E. Mergenhagen, 1970, J. Periodontol. 41, 71. Swanson, M.J. and E.L. Becker, 1976, J. Immunol. Methods 13, 191. Ward, P.A., C.G. Cochrane and H.J. Miiller-Eberhard, 1965, J. Exp. Med. 122, 327. Wilkinson, P.C., 1974, Chemotaxis and Inflammation (Churchill Livingstone, Edinburgh and London) pp. 42--45. Zigmond, S.H. and J.G. Hirsch, 1973, J. Exp. Med. 137,387.
