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A simple microscopic method for identifying and quantitating phagocytic cells in vitro
Journal oflmmunological Methods, 18 (1977) 377--379
377
© Elsevier/North-Holland Biomedical Press Short c o m m u n i c a t i o n A SIMPLE MICROSCOPIC METHOD F O R I D E N T I F Y I N G AND Q U A N T I T A T I N G PHAGOCYTIC CELLS IN V IT RO
JUDITH PATTERSON-DELAFIELD and ROBERT I. LEHRER Department of Bacteriology and Division of Hematology-Oncology, Department of Medicine, University of California, Los Angeles, CA 90024, U.S.A.
(Received 26 May 1977, accepted 8 June 1977)
A simple method is described for distinguishing phagocytic cells from non-phagocytic cells in a mixed population of leukocytes.
There is f r equent l y a need for rapid and quantitative assessment of the n u m b e r of phagocytic cells in mixed populations of leukocytes. We have developed a simple m e t h o d t ha t uses a stain to distinguish internalized from extracellular or attached particles. We believe others may find it similarly useful, and herein describe it. We used heat-killed, opsonized yeast cells, Candida albicans or Candida parapsilosis as particles for phagocytosis. The phagocytic cells we examined included human neutrophils and m o n o c y t e s and rabbit alveolar and peritoneal macrophages. Fungi were grown in Sabouraud's 2% dextrose brot h for 2--3 days at 33°C. T o prepare the yeast cells, 109 fungi were washed twice in distilled water, resuspended in water and heated 30 min at 90 ° C. T hey were washed again and resuspended in sterile distilled water. Such cells can be stored u n d er sterile conditions in the cold for at least t w o months. Opsonized particles were prepared by mixing a p p r o x i m a t e l y l 0 s Candida in 1 ml of specific rabbit antisera diluted 1 : 20 with fresh frozen normal h u m a n t y p e AB serum th at had been stored at --70°C. For studies with human leukocytes, the yeasts were opsonized with G r oup AB serum alone. The yeast and sera were incubated at 37°C for 30 min, t he n centrifuged and washed twice before resuspending t h e m at the desired c o n c e n t r a t i o n in Hanks' balanced salt solution (HBSS) (Grand Island Biological Com pany, Grand Island, NY). Rabbit alveolar and peritoneal macrophages were obtained by repeatedly lavaging rabbit lungs (Myrvik et al., 1961) or peritoneal cavities with heparinized (5 I.U./ml) Dulbecco's phosphate buffered saline, pH 7.4 (Grand Island Biological C o mp a ny, Grand Island, NY). Hum an p o l y m o r p h o n u c l e a r leukocytes and m o n o c y t e s were obtained f r om peripheral blood by standard and previously described m e t h o d s (Lehrer and Cline, 1969; Lehrer, 1972). In our experiments, the leukocytes were centrifuged and resuspended to a concen-
378 t r a t i o n o f 1 X 107/ml in HBSS w i t h 20% fetal calf s e r u m added. O u r usual i n c u b a t i o n m i x t u r e c o n s i s t e d o f 5 X 106 cells, 1 0 - - 2 0 X 106 y e a s t in 1 m l o f H B S S w i t h 20% fetal calf s e r u m . T h e m i x t u r e was r o t a t e d e n d - o v e r - e n d a t 3 7 ° C a n d s a m p l e d at intervals. A f e w d r o p s o f s a m p l e w e r e m i x e d w i t h an e q u a l v o l u m e o f a s o l u t i o n c o n t a i n i n g 0.4% t r y p a n blue and 0.2% eosin Y in p h o s p h a t e b u f f e r e d saline. Wet m o u n t s w e r e o b s e r v e d b y d i r e c t m i c r o s c o p y . As t h e i n t e r n a l i z e d y e a s t s w e r e p r o t e c t e d f r o m the d y e s t h e y r e m a i n e d u n s t a i n e d , b u t w e r e clearly visible d u e t o t h e i r large size and c h a r a c t e r i s t i c shape. T h e free or s u r f a c e - a d h e r e n t y e a s t s w e r e stained p u r p l e a n d w e r e easily distinguished f r o m t h e p h a g o c y t i z e d y e a s t (fig. 1). When an excess o f particles is used, t h e t o t a l p e r c e n t a g e o f p h a g o c y t i c cells is readily d e t e r m i n e d . In a d d i t i o n , t h e p e r c e n t a g e o f n o n - v i a b l e l e u k o c y t e s can be e s t i m a t e d s i m u l t a n e o u s l y , b y o b s e r v i n g t h e p e r c e n t a g e o f l e u k o c y t e s perm e a b l e t o t h e stain (inset o f fig. 1). We believe t h a t this simple p r o c e d u r e p e r m i t s c a l c u l a t i o n s o f p h a g o c y t i c
Fig. 1. Rabbit alveolar macrophages and Candida albicans. Heat-killed yeast cells and macrophages were incubated as described in the text. Intracellular yeasts, indicated by the arrows, are unstained. Three extracellular yeasts, darkly stained, are adjacent to the lower macrophage. The inset shows nuclear and cytoplasmic staining of a non-viable macrophage. Original magnification × 2500.
379 indices m o r e precisely t h a n o t h e r m e t h o d s , a n d will be useful in d e t e r m i n i n g t h e f u n c t i o n a l characteristics o f cell p o p u l a t i o n s c o n t a i n i n g p h a g o c y t e s . REFERENCES Lehrer, R.I., 1972, J. Clin. Invest. 51, 2566. Lehrer, R.I. and M.J. Cline, 1969, J. Bacteriol. 98,966. Myrvik, Q.N., E.S. Leake and B. Fariss, 1961, J. Immunol. 86, 128. This work was supported, in part, by grants from the U.S. Public Health Service, AI 12171 and CA 15487, and by funds from the California Institute for Cancer Research.
377
© Elsevier/North-Holland Biomedical Press Short c o m m u n i c a t i o n A SIMPLE MICROSCOPIC METHOD F O R I D E N T I F Y I N G AND Q U A N T I T A T I N G PHAGOCYTIC CELLS IN V IT RO
JUDITH PATTERSON-DELAFIELD and ROBERT I. LEHRER Department of Bacteriology and Division of Hematology-Oncology, Department of Medicine, University of California, Los Angeles, CA 90024, U.S.A.
(Received 26 May 1977, accepted 8 June 1977)
A simple method is described for distinguishing phagocytic cells from non-phagocytic cells in a mixed population of leukocytes.
There is f r equent l y a need for rapid and quantitative assessment of the n u m b e r of phagocytic cells in mixed populations of leukocytes. We have developed a simple m e t h o d t ha t uses a stain to distinguish internalized from extracellular or attached particles. We believe others may find it similarly useful, and herein describe it. We used heat-killed, opsonized yeast cells, Candida albicans or Candida parapsilosis as particles for phagocytosis. The phagocytic cells we examined included human neutrophils and m o n o c y t e s and rabbit alveolar and peritoneal macrophages. Fungi were grown in Sabouraud's 2% dextrose brot h for 2--3 days at 33°C. T o prepare the yeast cells, 109 fungi were washed twice in distilled water, resuspended in water and heated 30 min at 90 ° C. T hey were washed again and resuspended in sterile distilled water. Such cells can be stored u n d er sterile conditions in the cold for at least t w o months. Opsonized particles were prepared by mixing a p p r o x i m a t e l y l 0 s Candida in 1 ml of specific rabbit antisera diluted 1 : 20 with fresh frozen normal h u m a n t y p e AB serum th at had been stored at --70°C. For studies with human leukocytes, the yeasts were opsonized with G r oup AB serum alone. The yeast and sera were incubated at 37°C for 30 min, t he n centrifuged and washed twice before resuspending t h e m at the desired c o n c e n t r a t i o n in Hanks' balanced salt solution (HBSS) (Grand Island Biological Com pany, Grand Island, NY). Rabbit alveolar and peritoneal macrophages were obtained by repeatedly lavaging rabbit lungs (Myrvik et al., 1961) or peritoneal cavities with heparinized (5 I.U./ml) Dulbecco's phosphate buffered saline, pH 7.4 (Grand Island Biological C o mp a ny, Grand Island, NY). Hum an p o l y m o r p h o n u c l e a r leukocytes and m o n o c y t e s were obtained f r om peripheral blood by standard and previously described m e t h o d s (Lehrer and Cline, 1969; Lehrer, 1972). In our experiments, the leukocytes were centrifuged and resuspended to a concen-
378 t r a t i o n o f 1 X 107/ml in HBSS w i t h 20% fetal calf s e r u m added. O u r usual i n c u b a t i o n m i x t u r e c o n s i s t e d o f 5 X 106 cells, 1 0 - - 2 0 X 106 y e a s t in 1 m l o f H B S S w i t h 20% fetal calf s e r u m . T h e m i x t u r e was r o t a t e d e n d - o v e r - e n d a t 3 7 ° C a n d s a m p l e d at intervals. A f e w d r o p s o f s a m p l e w e r e m i x e d w i t h an e q u a l v o l u m e o f a s o l u t i o n c o n t a i n i n g 0.4% t r y p a n blue and 0.2% eosin Y in p h o s p h a t e b u f f e r e d saline. Wet m o u n t s w e r e o b s e r v e d b y d i r e c t m i c r o s c o p y . As t h e i n t e r n a l i z e d y e a s t s w e r e p r o t e c t e d f r o m the d y e s t h e y r e m a i n e d u n s t a i n e d , b u t w e r e clearly visible d u e t o t h e i r large size and c h a r a c t e r i s t i c shape. T h e free or s u r f a c e - a d h e r e n t y e a s t s w e r e stained p u r p l e a n d w e r e easily distinguished f r o m t h e p h a g o c y t i z e d y e a s t (fig. 1). When an excess o f particles is used, t h e t o t a l p e r c e n t a g e o f p h a g o c y t i c cells is readily d e t e r m i n e d . In a d d i t i o n , t h e p e r c e n t a g e o f n o n - v i a b l e l e u k o c y t e s can be e s t i m a t e d s i m u l t a n e o u s l y , b y o b s e r v i n g t h e p e r c e n t a g e o f l e u k o c y t e s perm e a b l e t o t h e stain (inset o f fig. 1). We believe t h a t this simple p r o c e d u r e p e r m i t s c a l c u l a t i o n s o f p h a g o c y t i c
Fig. 1. Rabbit alveolar macrophages and Candida albicans. Heat-killed yeast cells and macrophages were incubated as described in the text. Intracellular yeasts, indicated by the arrows, are unstained. Three extracellular yeasts, darkly stained, are adjacent to the lower macrophage. The inset shows nuclear and cytoplasmic staining of a non-viable macrophage. Original magnification × 2500.
379 indices m o r e precisely t h a n o t h e r m e t h o d s , a n d will be useful in d e t e r m i n i n g t h e f u n c t i o n a l characteristics o f cell p o p u l a t i o n s c o n t a i n i n g p h a g o c y t e s . REFERENCES Lehrer, R.I., 1972, J. Clin. Invest. 51, 2566. Lehrer, R.I. and M.J. Cline, 1969, J. Bacteriol. 98,966. Myrvik, Q.N., E.S. Leake and B. Fariss, 1961, J. Immunol. 86, 128. This work was supported, in part, by grants from the U.S. Public Health Service, AI 12171 and CA 15487, and by funds from the California Institute for Cancer Research.