Separation of round spermatids from the rat using an immunoselection panning technique.
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 x 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).
Pelengaris
S A
SA
Institute of Zoology, Zoological Society of London, United Kingdom.
Moore
H D
HD
D000818:Animals; D000911:Antibodies, Monoclonal; D002469:Cell Separation / Q000379:methods*; D007163:Immunosorbent Techniques; D008297:Male; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D013087:Spermatids / Q000166:cytology* / Q000276:immunology; D013737:Testis / Q000166:cytology
D000911:Antibodies, Monoclonal