F12 ham dmem f12k

bMECs were isolated from the alveolar tissue of the udders of healthy lactating cows as previously described [13 (link)]. Cells from passages 2–8 were used in all of the experiments. The bMECs were cultured in growth medium (GM) that was composed of a DMEM medium/nutrient mixture F12 Ham (DMEM/F12K, Sigma) supplemented with 10% fetal calf serum (Equitech Bio), 10 μg/mL insulin (Sigma), 5 μg/mL hydrocortisone (Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 μg/mL amphotericin B (Invitrogen). The cells were grown in a 5% CO₂ atmosphere at 37°C. To perform the E2 and/or S. aureus challenge, polarized monolayers of bMECs (dishes covered with 6–10 μg/cm² rat-tail type I collagen, Sigma) were cultured in serum-free DMEM/F12K without phenol red (Sigma) and antibiotics (incomplete medium) for 24 h, and then they were treated with the hormone and/or infected with the bacteria.

bMECs isolation was performed on udder alveolar tissue from healthy lactating cows as described (Anaya-López et al., 2006 (link)). Cells from passages 2–8 were used in all of the experiments. The cells were cultured in petri dishes (Corning-Costar) in growth medium (GM) that was composed of a DMEM medium/nutrient mixture F-12 Ham (DMEM/F-12K, Sigma), which was supplemented with 10% fetal calf serum (Equitech Bio), 10 μg/ml insulin (Sigma), 5 μg/ml hydrocortisone (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml amphotericin B (Invitrogen). The bMECs were grown in 5% CO₂ atmosphere at 37°C.