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Conclusion:
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Flow cytometry analysis showed that the relative abundances of undifferentiated spermatogonia (SgU), expressing CDH1high, ITGA6high, cKITlow, and of differentiated spermatogonia (SgD), expressing CDH1low, ITGA6low, cKIThigh, were similar between Ctrl and DKO animals.
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Comments:
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Testicular cell suspension was prepared by incubation of seminiferous tubules in 200 U/ml Collagenase type I (Worthington Biochemical LS004196), 5 ?g/ml DNAse I (Roche 10104159001), and 0.05%Trypsin (Gibco 25200056) in GBSS (Sigma G9779) as described in (100). Cells were stained with 1/200 anti-CD117/c-kit PE (eBioscience 12-1171-83), anti-CD324/E-Cadherin eFluor 660 (eBioscience 50-3249-82) and anti-CD49f/Integrin alpha 6 PE-Cyanine7 (eBioscience 25-0459-82) for 1 hour at 32oC with constant shaking and protected from light. After washing cells were incubated with 20 ?g/ml with Hoechst 33342 (Thermo Fischer Scientific H3570) for 1 hour at 32oC with constant shaking and protected from light and then with 30nM DRAQ7 (Biostatus DR71000) for 5 minutes on bench. The stained cells were strained through a 40??m nylon filter into 5 ml polypropylene tubes and were sorted on a BD FACSAria III cell sorter fitted with a 70??m nozzle (Becton Dickinson) using a 375nm laser to excite Hoechst 33342, a 561nm laser to excite PE and PE-Cy7 and a 633nm laser to excite DRAQ7 and eFluor600. Cells were first gated for FSC and SSC to exclude debris. Then the live cells were selected based on the absence of DRAQ7 signal detected using 755LP, 780/60BP. Then we gated for cells positive for Hoechst 33342 emission which was detected using a 670LP (Hoechst-Red) and 450/20 BP (Hoechst-Blue). We gated for the Hoechst-Redlow and Hoechst-Bluemid population which is enriched for 2N spermatogonia. Fluorescence for PE was detected using a 582/15BP filter, for PE-Cy7 was detected using 735LP,780/60BP filter and for eFluor660 using a 660/20BP filter. Undifferentiated spermatogonia were sorted as CD324high, CD49fhigh, CD117low and differentiated spermatogonia were sorted as CD324low, CD49flow, CD117high.
For sperm cell sorting, mouse cauda epididymides were dissected into a Petri dish and fat patches were removed with forceps and scissors. Each epididymis was punctured with a needle and carefully squeezed into 100 ul of PBS with the help of two forceps. Sperm cell suspension was transferred into an Eppendorf tube and was allowed to liquefy at room temperature for 5 minutes. To break sperm tails, cells were briefly sonicated with a Brandson Tip digital sonicator with 10% amplitude and 3 cycles of 0.5 sec ON / 2 sec OFF. Cells were stained with 2 ul/ml Hoechst 33342 (H3570, ThermoFisher) for 1h at 25°C with constant shaking and protected from light. Cells were filtered through a 40 ?m Nylon filter into 5 ml polypropylene tubes prior to sorting into PBS with a BD FACS Aria III instrument. Cells were first gated for FSC and SSC to exclude debris. Then sperm cells were sorted according to their 1C DNA content.
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