The alternative procedure for using EZ-Flask is to fill with 250ml of your usual culture media, ideally with some T-Flask conditioned media, then inoculate 10 to 25 million cells. Wait 2 days then add 250 ml. Wait another 3 or 4 days then add 500 ml to take the total volume up to 1 Litre. On day 30 harvest 1 Litre mAb.