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  • Acrosome Biogenesis in the Hamster: MATERIALS AND METHODS(2)

Spermatogenic Cell Isolation

Spermatogenic cells were purified by unit gravity sedimentation . Testes were minced and rinsed twice in Krebs-Ringer-bicarbonate (KRB) medium containing both nonessential and essential amino acids. The tissue was then incubated for 30 min at 37°C in KRB containing 1 mg/ml collagenase (Sigma Chemical Co.) with gentle oscillation and then incubated for 10 min in KRB containing 1 mg/ml trypsin and 0.1 U/ml micrococcal nuclease (Sigma Chemical Co.). buy ortho tri-cyclen online

After 3 rinses with KRB, the seminiferous tubule fragments were resuspended in KRB containing 0.5% BSA, gently pipetted, and filtered through a 40-(jlM mesh sieve (Falcon 2340; Falcon Plastics, Los Angeles, CA) to obtain a suspension of single cells. Cells were counted and assessed for viability by Trypan blue dye exclusion, and samples exhibiting > 90% viability were used for unit gravity sedimentation. Cells were separated for 2 h at 4°C on a 24% gradient of BSA in KRB medium using a Celsep apparatus (Brinkman Instruments, Westbury, NY). Purity of gradient fractions was determined by phase contrast and/or differential interference contrast (DIC) microscopy. Fractions containing > 80% round spermatids were pooled, and the cells were pelleted by centrifugation for 10 min at 500 X g-

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