622 



H. MOSER, I. HADJI-AZIMI AND S. SLATKINE 



Methods of cell dispersion 



A. Adulî and advanced larxal tissues 



Cell suspensions derived from internai oigans (for example kidney) of frogs 

 and of large tadpoles are prepared by treating tissues with our standard cell- 

 dispersion médium CDM-A1 which contains 0.5% trypsin in Ca ++ and Mg~ T 

 free Barth-electrolyte solution. D( — )-glucose, bovine albumin and antibiotics 

 (see Table III). Decontaminated adults or larvae are transfered into stérile oper- 

 ating dishes, dissected with stérile forceps and scissors, and the isolated organs 

 are washed twice in stérile wash-medium WS-AO (see Table II). The tissue is 

 eut into small pièces in a Pétri dish containing trypsinization médium CDM-A1. 

 Subsequently the tissue fragments are transfered into a pyrex test tube and over- 

 laid with 5 ml of fresh cell-dispersion médium. Trypsinization is carried eut at 

 room température with occasional manual agitation, the trypsin solution being 

 replaced with fresh solution at intervals of 20 minutes. With each média change 

 the supernatant cell suspension is collected and introduced into a flask to which 

 has been added previously, in order to inhibit further action of trypsin upon 

 isolated cells, 10 ml of complète growth médium (TCM-A2). Finally the pooled 

 cell suspension including the remaining undigested tissue fragments are centri- 

 fuged for 10 minutes at 112 G (1000 rpm at a radius of 10 cm), and the cells 

 resuspended in fresh growth médium. The harvest of cells is planted into culture 

 dishes or vessels in aliquots of a few hundred thousand cells per 4 to 5 ml liquid 

 médium. 



Table II 



Composition of Ca^~ and Mg ^ ~ free wash-solutions for Xenopus laevis cells and t issues 



Wash solutions (1000 ml) 



WS-AO 



WS-A3 



+ 



+ X 

 tùb- 



1% 



NaCl 



KCL 



Na ,HP0 4 12 H„0 . 



KH.,P0 4 



NaHC0 3 .... 



H 2 



Penicillin G cryst . 

 Streptomycin sulfate 

 Tetracvcline-HCl . 



6.800 grams 

 0.100 grams 

 0.277 grams 

 0.020 grams 

 0.200 grams 

 1000 ml 

 0.000 unit s 

 0.000 grams 

 0.000 arams 



6.800 grams 

 0.100 grams 

 0.277 grams 

 0.020 grams 

 0.200 grams 

 1000 ml 

 300,000 units 

 300 mg 

 10 mg 



WS-AO: Sterilized by autoclaving for 30 minutes at 121' C; stored at room température in 100 ml aliquots 

 in screw capped bottles. 



WS-A3: Sterilized by filtration through Millipore filter. type GS, 0.22 -i. pore size. under positive pressure. 

 Stored frozen at — 25 C in 100 ml aliquots in screw capped bottles. until used. 



