Isotope uptake of individual cells: Technique for stained autoradiographs and microphotographs for grain counting

Abstract

1 dt N or er o investigate the uptake of radioactive isotopes in bone marrow cultures it was essential to devise a method for determining the uptake and localisation in individual cells. The technical difficulties of localising a counter over a particular cell are great, and usually the amount of radioactivity per cell is too small for direct detection with such counters. The use of autoradiography eliminates these difficulties. The best resolution is given by the stripping film technique (1, 3), but in order to eliminate as far as possible back scatter from the slide and lateral radiation from neighbouring cells it is important that the distance in between the cells should be at least twice the diameter of the cells. Bone marrow or blood cell suspensions cultured in vitro (2) yield smear preparations which fulfil these requirements. The morphology of the individual cells is well preserved, they are in a monocellular layer, and are suitably far apart. For the work planned it was essential to be able to perform accurate differential counts on the autoradiographs, and it was therefore important to be able to stain the cells with one of the Romanowsky stains. Phase contrast examination, though giving valuable information about cytological details, will not allow differentiation of all cell types of the bone marrow with certainty. Staining of the smears before coating them with the stripping film is contraindicated. Not only is there a possiblility of removal of isotope from the cells during the staining process, but also the stain might produce artefacts in the photographic emulsion during the time of exposure, and finally the development and fixation might alter the staining very adversely. It has been found, however, that if the autoradiographs are carefully washed in running cold water for 4-6 hours after processing, and the optimal pH (6.8) is maintained by using buffered distilled water, then one hours staining with a 1 : 12 dilution of a Leishman-Giemsa mixture (1 part Leishman solution and 3 part Giemsa solution) gives as good staining as any ordinary smear. The procedure is:1. Make smears on slides, fix smears for l-2 mins in 95 per cent alcohol. 2. Coat smears with stripping film as described by Pelt (1, 3). 3. After exposure develop 4 mins. in Kodak D. 19b, fix for 15-18 minutes in half strength fixing solution. 4. Wash 2-4 hours in running cold tap water. 5. Stand for 2 hours