Antenatal diagnosis of genetic disease / edited by A.E.H. Emery.

Date:
1973
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Antenatal diagnosis of genetic disease / edited by A.E.H. Emery. Source: Wellcome Collection.

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    APPENDIX 159 Decant supernatant all but 1 ml and resuspend cells in tliis. Divide cells between number of new dishes required. Add appropriate amount of medium to new dishes, incubate and medium change at 3 days. (N.B. Cell counts on the cell suspension after trypsinization will allow the optimum inoculum required to be estimated for each new dish.) Subculture method for small quantities of cells Wash the culture dish twice at least in Dulbecco solution (phosphate-buffered saline, Dulbecco A). Add 2 to 5 ml trypsin (0-25 per cent trypsin in Dulbecco A). Swirl the trypsin over the culture and then remove as much as possible, leaving only a film. Incubate for 15 to 30 min till cells are detached. Add medium to correct amount for the size of the dish if the aim is to re¬ distribute the cells in the original culture dish. If new dishes are to be set up, add sufficient medium to resuspend the cells and divide between the required number of new dishes. Chromosome Preparations Coverslip chromosome preparations When cells are almost confluent and are noted to be in mitosis with light microscopy or phase, add colchicine to dish to give final concentration of 1 /40,000 (w/v). Incubate for hours. Remove medium and replace with hypotonic solution (0-075 м-KCl). Leave at room temperature for 50 min.) Remove half the hypotonic solution and replace with fixative (methanol+ acetic acid, 3:1). Leave for 5 min. Repeat twice more. Finally remove all mixture and replace with fixative only. Leave 10 min. Air or flame dry. Stain with appropriate stain, e.g. Giemsa—10 min in 10 per cent Giemsa; or Atebrin—as for Y bodies. Rinse in tap water, dehydrate in acetone and clear in xylene. Cell suspension for chromosome studies When cells are ready for examination, remove medium and trypsinize as for subculture. Spin cell suspension and decant trypsin-medium mixture. Resuspend cells in hypotonic solution (0-075 м-KCl). Leave for 30 min at room temperature. Spin for 5 minutes at 800 to 900 rev/min. Decant hypotonic solution and resuspend cells in 2 mis fixative (methanoH-acetic acid, 3:1). Stand for 30 min. Spin for 5 min and resuspend in 0-25 ml fixative. The cells are now ready for use and may be stored at 4°C. Stain as required. Metachromasia When growth is confluent enough for study, remove cover-slip from culture dish. Wash well in normal saline. Fix cells by either of the following methods: (a) Leave for 30 min in the following fixative : 10 ml Pb NO3 2 per cent solution 10 ml formalin 80 ml ethanol 100 ml (N.B. the solution may turn cloudy when poured on—agitate gently.) Wash in 70 per cent, 80 per cent and absolute ethanol. Air dry. (b) Leave for 10 min. in methanol. Air dry.