Abstract
Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use.
Highlights
Spermatogenesis is a complex differentiation process essential for all the species with sexual reproduction, which leads to the formation of male gametes
Spermatogenesis can be divided into three phases: mitotic proliferation of spermatogonia, meiotic divisions of spermatocytes, and spermiogenesis
A strategy used to allow the analysis of gene expression along spermatogenesis has been the use of whole testes of prepubertal specimens, naturally enriched in early spermatogenic stages [4, 5]
Summary
Spermatogenesis is a complex differentiation process essential for all the species with sexual reproduction, which leads to the formation of male gametes. A strategy used to allow the analysis of gene expression along spermatogenesis has been the use of whole testes of prepubertal specimens, naturally enriched in early spermatogenic stages [4, 5] This approach does not precisely allow the assignment of specific transcripts to individual cell types. Methods involving a combination of gentle mechanical action with enzymatic treatment have rendered better results in terms of yield and cell type representation, while minimizing cell aggregation [3, 9]. We present a very simple and efficient protocol to rapidly obtain a cellular suspension from testis material for flow cytometric analysis This protocol eliminates steps between animal sacrifice and spermatogenic stage-specific molecular studies, aiming to optimize macromolecule preservation
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