Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.