Separation of round spermatids from the rat using an immunoselection panning technique
✍ Scribed by S. A. Pelengaris; Professor H. D. M. Moore
- Publisher
- John Wiley and Sons
- Year
- 1995
- Tongue
- English
- Weight
- 568 KB
- Volume
- 41
- Category
- Article
- ISSN
- 1040-452X
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✦ Synopsis
Abstract
A method was devised for the isolation of round spermatids from the rat using a positive immunoselection technique (panning). A testis suspension was prepared from adult rats by enzymatic digestion of seminiferous tubules with collagenase. Specific mouse monoclonal antibody (97.25) was indirectly attached to Petri dishes and used in a panning protocol to purify spermatids from the testis cell suspension. The quantity and purity of cells isolated were determined by cell counts and histochemical (periodic acid‐Schiff stain) or by immunostaining with acrosome‐specific antibodies. A mean yield of 1.38 ± 0.15 × 10^7^ cells per dish was obtained with a purity of more than 90%. The viability of the cells was confirmed by epifluorescent microscopy with propidium iodide/carboxyfluorescein acetate probes. Northern blot analysis of RNA extracted directly from the dish indicated good integrity of a spermatid‐specific transcript of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). © 1995 Wiley‐Liss, Inc.