Buffalo MECs primary culture and heat treatment

The buffalo mammary gland tissues of approximately 5gm were obtained from a healthy adult buffalo from Gazipur abattoir 28.734190N and 77.272830E, New Delhi, India. The primary MECs were cultured using DMEM/F12, supplements and growth conditions as described earlier [13]. After several passages, 80% confluent buffalo MECs on 10th passage were distributed in collagen treated 12-wellplates (Corning, USA) in two sets with one plate assigned as control (kept at 37°C all the time) and other plate as treated (exposed to 42°C). Initially, cells were incubated at 37°C with 5% CO₂ to stabilize the culture. Subsequently, the plate marked as treated was exposed to 42°C for one hour to simulate heat stress (HS) condition. After 1h, the cells were allowed to recover at 37°C, 5% CO₂and harvested by trypsinization at different time points (30m, 2h, 4h, 8h, 12h, and 24h). The samples from control (CTR) plates were also trypsinized and harvested at the same time points corresponding to the treated plates. Followed by exposure to heat stress, cell viability and growth characteristics of buffalo MECs in normal and heat treated samples were determined using commonly used trypan blue dye exclusion method.