Defrosting / waking up mammalian cells, written by Rosa Schier

• Keep the cells on dry ice, from the -80°C fridge or the liquid nitrogen tank
• Prepare a 15ml tube with 10ml of new media (corresponding to the cells) under the hood
• Defrost the cells by holding them into a water bath at 37°C, don’t defrost the cells completely so they don’t get damaged (keep a little ice cube). When you return under the hood attention to disinfect the tube correctly.
• Transfer the approximately 1ml of cells into the 15ml tube prepared before, mix with the pipette (5ml) and invert the tube 2-3 times (carefully)
• Take a sample to count the cells
• Centrifuge the cells 3min at 1000rpm. During this time calculate the volume needed to resuspend in function of the number of cells you have. The concentration should be around 0.45x106cell/ml
• Vacuum the media and resuspend the cells in new media which volume was calculated before
• If the cells are not used to agitation, transfer them into a T-flask and put at 37°C (once the cells grew a bit you can try to put them in suspension)
• If the cells are in suspension, transfer them depending on the volume in a 50ml tube, a maxi spin tube or an erlenmeyer and put them in agitation at 37°C