Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis
Tóm tắt
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.
Tài liệu tham khảo
Gilbert SF: Developmental Biology. 1991, Sunderland, Massachusettts, Sinauer Associates, 3
Rice WR, Chippindale AK: Sexual recombination and the power of natural selection. Science. 2001, 294: 555-559. 10.1126/science.1061380.
Meistrich ML: Separation of spermatogenic cells and nuclei from rodent testes. Methods of Cell Biology. Edited by: Prescott DM. 1977, Academic, New York, 15: 15-54. 10.1016/S0091-679X(08)60207-1.
Thomas KH, Wilkie TM, Tomashefsky P, Bellvé AR, Simon MI: Differential gene expression during spermatogenesis. Biol Reprod. 1989, 41: 729-739. 10.1095/biolreprod41.4.729.
Almstrup K, Nielsen JE, Hansen MA, Tanaka M, Skakkebæk NE, Leffers H: Analysis of cell-type-specific gene expression during mouse spermatogenesis. Biol Reprod. 2004, 70: 1751-1761. 10.1095/biolreprod.103.026575.
Kotaja N, Kimmins S, Brancorsini S, Hentsch D, Vonesch JL, Davidson I, Parvinen M, Sassone-Corsi P: Preparation, isolation and characterization of stage-specific spermatogenic cells for cellular and molecular analysis. Nat Methods. 2004, 1: 249-254. 10.1038/nmeth1204-249.
Lam DMK, Furrer R, Bruce WR: The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse. Proc Natl Acad Sci USA. 1970, 65: 192-199. 10.1073/pnas.65.1.192.
Romrell LJ, Bellve AR, Fawcet DW: Separation of mouse spermatogenic cells by sedimentation velocity. Dev Biol. 1976, 19: 119-131. 10.1016/0012-1606(76)90262-1.
Malkov M, Fisher Y, Don J: Developmental schedule of the postnatal rat testis determined by flow cytometry. Biol Rep. 1998, 59: 84-92. 10.1095/biolreprod59.1.84.
Aslam H, Schneiders A, Perret M, Weinbauer GF, Hodges JK: Quantitative assessment of testicular germ cell production and kinematic and morphometric parameters of ejaculated spermatozoa in the grey lemur, Microebus murinus. Reproduction. 2002, 123: 323-332. 10.1530/rep.0.1230323.
Vigodner M, Lewin LM, Shochat L, Mittelman L, Golan R: Meiosis in the golden hamster: a confocal microscopy and flow cytometric analysis. Molec Reprod Dev. 2003, 64: 86-95. 10.1002/mrd.10212.
Wistuba J, Schrod A, Greve B, Hodges JK, Aslam H, Weinbauer GF, Luetjens M: Organization of seminiferous epithelium in primates: relationship to spermatogenic efficiency, phylogeny, and mating system. Biol Reprod. 2003, 69: 582-591. 10.1095/biolreprod.103.015925.
Geisinger A: Spermatogenesis in Mammals: a very peculiar cell differentiation process. Cell Differentiation Research Developments. Edited by: Ivanova LB. 2008, Nova, New York, 97-123.
Meistrich ML: Separation of mouse spermatogenic cells by velocity sedimentation. J Cell Physiol. 1972, 80: 299-312. 10.1002/jcp.1040800218.
Meistrich ML, Bruce WR, Clermont Y: Cellular composition of fractions of mouse testis cells following velocity sedimentation separation. Exp Cell Res. 1973, 79: 213-227. 10.1016/0014-4827(73)90507-7.
Rosu F, Gabelica V, Houssier C, De Pauw E: Determination of affinity, stoichiometry and sequence selectivity of minor groove binder complexes with double-stranded oligodeoxynucleotides by electrospray ionization mass spectrometry. Nucleic Acids Res. 2002, 30: e82-10.1093/nar/gnf081.
Rodriguez R, Wettstein R: Quantitative study on guinea pig spermatogenesis shows a relative high percentage of early meiotic prophase stages. Anat Rec. 2004, 278: 493-504. 10.1002/ar.a.20037.