Identification of Sertoli cells and germ cells in co-cultures of PS with Sertoli cells on day 3, by using a monoclonal antibody against vimentin conjugated with a red fluorochrome, Alexa 647.

<p>a: Representative flow cytometry analysis of co-cultured cell populations on day 3: Distribution of vimentin-positive diploid Sertoli cells (R3), vimentin-positive tetraploid Sertoli cells (R4), and vimentin-negative germ cells (R2) according to their ploidy (Hoechst 33342). The high specificity of the anti-vimentin antibody was shown by the near complete disappearance of R3 and R4 regions, when co-cultured cells were incubated with non immune mouse IgG conjugated with Alexa 647 as a control (b). Vimentin-positive diploid Sertoli cells (R3) and vimentin-positive tetraploid Sertoli cells (R4) were sorted on glass slides and examined for Hoechst (c, f) and vimentin (d, g) by fluorescent confocal microscopy. e and h are the transmission pictures corresponding respectively to c, d and f, g. Isolated Sertoli cells (c, d, e) and doublets of Sertoli cells (f, g, h) (as highlighted by the double blue spots showing the two nuclei labelled by Hoechst 33342) were identified in R3 and R4 regions respectively. All bars represent 10 µm.