Sperm-Oviduct Interaction: MATERIALS AND METHODS(2)

MATERIALS AND METHODS(2)

The media containing scraped tissues from ampulla and isthmus were collected separately and, after initial sedimentation, were gently centrifuged for 3 min at 100 X g. The supernatants were discarded, and 5 ml of medium A, containing tissue culture medium (TCM) 199 (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Life Technologies), 100 U/ml penicillin, 100 ^g/ml streptomycin, and 0.25 ^g/ml amphotericin B, was added to each pellet. The cells were mixed gently and were disaggregated by being passed once through a 21-gauge needle. The concentration of the epithelial cells was measured using a counting chamber. The viability was evaluated by mixing a sample of the cells with an equal volume of 4% Trypan blue (Sigma, Dorset, UK). Separate tissue culture flasks (75 cm2; Nalge Nunc International, Naperville, IL) were seeded with each preparation of isthmic and ampullar epithelial cells (1 X 106 viable epithelial cells/ ml). The flasks were incubated at 39°C in 100% humidity and 5% CO2 in air. The culture medium was refreshed every 48-72 h.

Epithelial cells reached confluency after 7-14 days. At confluency, cells were rinsed several times with PBS lacking Ca2+ and Mg2+ (Life Technologies) and were detached by incubating with 3 ml of trypsin-EDTA solution (Life Technologies) containing 0.5 mg/ml trypsin and 0.2 mg/ml EDTA at 39°C for 15-20 min. The viability and concentration of the detached cells were measured, and cells were suspended in TCM 199 supplemented with 10% FBS and 10% glycerol (BDH, Poole, UK) (1 X 106 viable cells/ml). The epithelial cell suspensions were divided into 1-ml portions in cryogenic vials (Nalge Nunc International), left overnight at — 70°C, and on the next day transferred to liquid nitrogen.

This entry was posted in Sperm and tagged Epithelial Cells, Oviduct, Sperm, Spermatozoa.