i need protocol for sertoli cell isolation procedure from rat. also for Germ cells. I wanted to do apoptosis studies. if any one knows help.
here are some links for your reference if you are not able to get them download, send me a message through PM i will mail you personally
Isolation of Sertoli Cells from Adult Rat Testes: An Approach to Ex Vivo Studies of Sertoli Cell Function www.biolreprod.org/cgi/rapidpdf/biolreprod.102.008045v1
A method for the isolation of intact Sertoli cell-germ cell associations from rat seminiferous tubules and their further partition into Sertoli cell and germ cell fractions
MEENAKUMARI and S. DURAISWAMI
Department of Zoology, University of Delhi, Delhi 110 007, India
Abstract: A technique is described for obtaining a Sertoli cell-enriched and a germ cell-enriched fraction from immature rat testes. Sertoli cell-germ cell associations were obtained by incubating washed seminiferous tubule fragments with ollagenase and Pancreatin. They were then manually dissociated into a suspension comprising Sertoli cells as well as the various germ cell types characteristic for a given day of ontogeny. Fractionation into a Sertoli cell-enriched fraction and a germ cell-enriched fraction was effected by centrifugation following layering over a stepwise gradient of Ficoll-400. While the time-span compares favourably with other procedures reported in the literature, it is believed this is the first time a method is described that enables the simultaneous recovery of both the Sertoli cells and the germ cells.
(J. Biosci., Vol. 10, Number 3, September 1986, pp. 413-422. © Printed in India.)
A rapid method of sertoli cell isolation by DSA lectin , allowing mitotic analyses
Department of Histology and Medical Embryology, University of Rome La Sapienza, Italy.
Abstract : We have developed a rapid and convenient method of Sertoli cell preparation for studying the growth kinetics of these cells in in vitro culture. Datura Stramonium agglutinin (DSA)-coated dishes were used to rapidly purify single Sertoli cells from immature rat testis. We have monitored by immunohistochemical markers the degree of contamination of our Sertoli cell preparation by other cell types. The cell preparation is essentially free of germ cells and interstitial cells and contains a minimal percentage of myoid cells. Sertoli cells isolated with this method retain functional activities such as the FSH responsiveness in terms of cAMP production. In addition, we have studied the proliferative activity of Sertoli cells isolated by lectin binding from rats of different ages. Sertoli cells exhibited a characteristic pattern of proliferation which was a function of the donor animal age. The proliferative activity of isolated Sertoli cells decreased with age, being much higher in 3 day-old rats than in older animals. A similar pattern was observed when the mitotic activity of Sertoli cells in response to mitogens present in the testicular extracts from 5 day-old rats was evaluated. The method described here reduces or eliminates many of the drawbacks of the conventional procedures used to isolate Sertoli cells, thus providing a useful tool in studies of growth kinetics and regulation of cell proliferation in vitro .
Mol Cell Endocrinol. 1998 Nov 25; 146(1-2):121-7