Journal of Immunological Methods, 22 (1978) 383--384 © Elsevier/North-Holland Biomedical Press



AHMED A. WADEE and RUBEN SHER Department of Immunology, School of Pathology of the University of Witwatersrand, and South African Institute for Medical Research, Johannesburg, South Africa

(Received 20 March 1978, accepted 26 March 1978) A fast and reproducible method for the staining of eosinophits on millipore filters for the study of chemotaxis is described. This procedure allows for the easy differentiation of eosinophils from other granulocytes both at the intra-filter level and on the filter surface. In vitro studies of eosinophil chemotaxis using millipore filters have been previously described (Kay, 1970; Clark et al., 1977). These studies have been carried o u t mainly on mixed populations of granulocytes containing various p r o p o r t i o n s o f eosinophils and neutrophils. In our experience, the techniques e m p l o y e d f or staining eosinophils are time consuming and result in technical problems, of t en making it difficult to differentiate eosinophils from o th er granulocytes with certainty. Firstly, the filter itself absorbs eosin dye mo r e quickly and to a greater e x t e n t than the eosinophil granules. Secondly, dehydrating the filters in increasing concentrations of ethanol results in a greater and m or e rapid loss of eosin from the granules than from the filter. To overcome these problems we have a d o p t e d a staining procedure which allows for optimal staining of the eosinophil granules and minimal staining o f the filters. Eosinophils were isolated according to the m e t h o d of Sher and Glover (1976). 90--95% purity was obtained and chemotaxis was measured using a modified B o y d e n c h a m b e r (Boyden, 1962) in which cells were separated f r o m the l e u c o a t t r a c t a n t by an 8 pm pore size m e m b r a n e filter (Millipore Corp., MA, U.S.A.). Chambers were incubated at 37°C for 4 h to record transfilter m o v e m e n t , or for 40 min according to the m e t h o d of Zigmond and Hirsch (1973), to measure intrafilter p enet rat i on achieved by the cells. L e u c o c y t e s were fixed by soaking the filter in m et hanol G.P. (Merck) for 5 sec, stained with 1% eosin (Fluka AG, Buchs SG, Switzerland) for 1.5 min and washed by dipping the filter in and out of water a few times. Cells were counterstained with h a e m a t o x y l i n (Ortho Diagnostics, G.R. Merck) for 30 sec, washed again, decolourised and d e h y d r a t e d in 70% ethanol for 1 min, followed by 5 sec in 100% ethanol. Excess ethanol was removed by dabbing the filter on a piece of blotting paper. The filters were placed on a slide, allowed to dry c om pl et el y and cleared with a drop of xyl ene prior to


Fig. 1. Stained eosinophils on a millipore filter.

microscopic examination. A representative example of eosinophil staining is shown in Fig. 1. The use of h a e m a t o x y l i n helps in differentiating between the two types o f granulocytes on the basis of their nuclei. Particular merits of the procedure we have described are t hat it allows sufficient staining time for the eosinophil granules to absorb eosin and a short destaining period facilitating m a x i m u m dyeloss from the filter. It differentiates the t w o major granulocyte populations from each ot her by the presence o f eosin-staining granules. T he technique is fast, reliable and can be applied to the observation of eosinophils b o t h on, or within the filter. Moreover, these filters can be kept for long periods when m o u n t e d on glass slides. REFERENCES Boyden, S., 1962, J. Exp. Med. 115,453. Clark, R.A.F., J.A. Sandier, J.I. Gallin and A.P. Kaplan, 1977, J. Immunol. 118,137. Kay, A.B., 1970, Clin. Exp. Immunol. 7,723. Sher, R. and A. Glover, 1976, Immunology 31,337. Zigmond, S.H. and J.G. Hirsch, 1973, J. Exp. Med. 137,387.

Staining of eosinophil granulocytes in chemotactic studies.

Journal of Immunological Methods, 22 (1978) 383--384 © Elsevier/North-Holland Biomedical Press 383 S h o r t communication S T A I NI NG OF EOSINOPH...
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