CULTURE OF CELLS AND TISSUES DERIVED FROM XENOPUS LAEVIS 



623 



B. Embryonic and young larval t issue s 



The continuous cell dispersion methods applied to adult Xenopus laevis 

 tissues do not yield satisfactory results with the larger and more fragile cells of 

 embryos and young ready-to-feed tadpoles. Therefore, cell suspensions derived 

 from whole embryos (stages 35 to 40) and from whole young swimming tadpoles 

 (stages 42 to 45) are prepared as follows: Aseptically reared embryos or larvae 

 are minced into small pièces (usually in numbers of 7-10) and the material trans- 

 fered into a pyrex test tube containing 5 ml of cell-dispersion médium. In the 

 case of advanced embryos the cell-dispersion médium contains either 0.017% 

 versene (cell dispersion médium CDM-A2, see Table III) or a mixture of 0.01% 

 versene and 0.2% trypsin (cell dispersion médium CDM-A3, see Table III); 

 in the case of young swimming tadpoles the standard cell dispersion médium 

 containing 0.5% trypsin (CDM-A1) is used. Cell dispersion is carried out at 

 room tempérai uie for a fixed period of 2 hours without agitation. Thereafter 

 the isolated cells and the remaining undigested tissue fragments are harvested by 

 jcentrifugation at 112 G for 10 minutes, resuspended in 6 ml of growth médium 

 TCM-A2, and aliquots of the cell suspension (usually 2 ml) seeded into culture 

 vessels. 



Table III 



Composition of cell-dispersion média for Xenopus laevis 





Cell-dispersion média (1000 ml) 



CDM-A1 



CDM-A2 



CDM-A3 



NaCl 



6.80 grams 



6.80 grams 



6.80 grams 



KC1 



0.10 grams 



0.10 grams 



0.10 grams 



Na 2 HP0 4 .12 H 2 . . . 



0.30 grams 



0.30 grams 



0.30 grams 



KH 2 P0 4 



0.02 grams 



0.02 grams 



0.02 grams 



NaHC0 3 



0.20 grams 



0.20 grams 



0.20 grams 



D(+)-glucose 



1.00 grams 



1.00 grams 



1.00 grams 



Bovine albumin 









fraction V (powder) . 



1.50 grams 



0.00 grams 



0.60 grams 



Versene 1 



0.00 grams 



0.17 grams 



0.10 grams 



Trypsin, Difco 1 :250 . . 



5.00 grams 



0.00 grams 



2.00 grams 



Phénol red solution, 1 % 2 



1 ml 



1 ml 



1 ml 



Penicillin G cryst . . . 



300,000 units 



300,000 units 



300,000 units 



Streptomycin sulfate . . 



300 mg 



300 mg 



300 mg 



Tetracycline-HCl . . . 



10 mg 



10 mg 



10 mg 



H a O 



999 ml 



999 ml 



999 ml 



Sterilized by fîltration through Millipore filter, type GS, 0.22 u, pore size, under positive pressure. Stored frozen 

 in 50 to 100 ml aliquots in screw capped bottles at — 25° C, until used. 



1 Ethylene diamine tetra acetic acid — Na 2 . 



2 DIFCO TC Phénol Red solution. 



