Establishment of Isthmic, Ampullar, and Porcine Kidney Epithelial Cell Culture in Chamber Slides
Frozen cryogenic vials of isthmic, ampullar, and LLC-PK1 (European Collection of Animal Cell Cultures, Wiltshire, UK) cells were thawed in a water bath at 37°C. Medium A (9 ml) was added to each cell type and mixed gently. The epithelial cells were centrifuged at 100 X g for 2 min. The supernatant was discarded, and a further portion of medium A (9 ml) was added to each cell sample. Cell concentration was adjusted to 5 X 104 viable cells/ml. Three wells of 4-well chamber slides (Lab-Tek II; Nalge Nunc International) were seeded with 800 ^l of isthmic, ampullar, and LLC-PK1 cell suspensions (4 X 104 viable cells). The chamber slides were incubated at 39°C in 100% humidity and 5% CO2 in air. The culture medium was refreshed every 48-72 h until confluency.
Boar semen diluted and stored for 24 h in Beltsville thawing solution was obtained from a commercial artificial insemination station (JSR Healthbred Limited, Thorpe Willoughby, Yorkshire, UK). Semen (10 ml) was washed three times with PBS by centrifugation and resuspension (600 Xg for 10 min). After the last centrifugation the supernatant was discarded, and the pellet was resuspended in a modified Tyrode’s medium ) containing 2 mM CaCl2, 3.1 mM KCl, 0.4 mM MgCl26H2O, 100 mM NaCl, 25 mM NaHCO3, 0.3 mM NaH2PO42H2O, 21.6 mM sodium lactate, 10 mM Hepes, 1 mM sodium pyruvate, and 6 mg/ml BSA.