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Immunomagnetic isolation of fetal rat gonocytes
R van den Ham1, A M van Pelt, M P de Miguel
1Department of Functional Morphology, Veterinary School, Utrecht University, The Netherlands.
Problem:
An efficient method to obtain highly enriched populations of viable gonocytes from rat embryos at Day 18 and Day 20 postcoitum (pc) is described.
Method:
Single-cell suspensions with high cell yield were obtained by a collagenase/ trypsin digestion of the decapsulated testis. The gonocytes were purified by a direct immunoseparation technique, using magnetizable beads coated with rat anti-mouse immunoglobulin M (IgM) and a monoclonal antibody 4B6.3E10, which specifically reacted with a differentiation antigen on the fetal germ cells.
Results:
Populations of 8.3 +/- 2.7 (x10(3); 18 days pc) or 1.2 +/- 0.25 (x10(4); 20 days pc) viable gonocytes per testis with purities of 91 +/- 6.5% and 92 +/- 4.3%, respectively, as determined by Nomarski microscopy were obtained.
Conclusion:
The cells were successfully used for culture studies and as starting material for the investigation of gene expression.