Culture of adherent cell lines Sample Clauses

Culture of adherent cell lines. All adherent cell lines were grown routinely in 10cm dishes or 75cm2 flasks depending on the number of cells needed for a given series of experiments. Cells were usually seeded at 106 cells in 12mls or 25mls of complete media+10% FCS depending on the culture vessel. Cells were grown at 37⁰C in a 5% CO2 humidified incubator (hereon referred to as 37⁰C/5%) for the indicated time or until 90-100% confluent. If cultured from a low density for an extended period (such as following thawing of limited number of cells) culture medium was changed every 3-4 days. Once cells reached an appropriately level of confluency they were passaged by removing media and incubating cells with 2-5mls of TrypLE™ express detachment reagent (preheated to 37⁰C) for ≈5 mins or until cells were detached. Cells were then washed in complete media to neutralise the trypsin analogue reagent. Following centrifugation cells were counted, resuspended in the appropriate volume of complete media, and seeded as described.