A modified method for spermatogonial cell sorting

Objective To improve the method of sorting undifferentiated and differentiated spermatogonial cells by magnetic bead sorting with specific antibodies. Methods Using the magnetic bead sorting technique combined with Thy1 and c-Kit specific antibodies, we sorted Thy1+ and c-Kit+ cells in the testis of 7-postnatal-day male mice as undifferentiated and differentiated spermatogonia, respectively. We determined the purities of the two types of spermatogonial cells by immunofluorescence and flow cytometry, identified them via the differential expressions of Gfrα1, Plzf, c-Kit and Sohlh2 by real-time quantitative PCR, and cultured the Thy1+ cells primarily. Results The purities of the Thy1+ and c-kit+ cells were as high as (85.65 ± 8.35)% and (89.40 ± 2.77)%, respectively (P Conclusions The magnetic bead sorting technique with Thy1 and c-Kit specific antibodies can be used to effectively identify undifferentiated and differentiated spermatogonia and culture undifferentiated Thy1+ cells in vitro.