<p>Flint Group Protocols</p><p>6. Reviving a cell line stored in liquid nitrogen</p><p>Defrosting cells.</p><p>The liquid nitrogen (LN) tank is located in an upstairs room. Use Blue Gloves when taking out samples. </p><p>1) Take off the lid of the LN tank. 2) Press the “Fill/Start” button. 3) Wait for the vapour to dissipate. 4) Lift out the rack of interest and remove sample tubes required from sample box. 5) Return rack to the tank. 6) Press the “Fill/Stop” button. 7) Replace the lid on the tank.</p><p>Transferring Neuro2A cells to a fresh TC flask.</p><p>1) Remember to warm a bottle of Minimum Essential Medium Eagle (MEME) in the water bath. It is kept in the fridge. Remember to ethanol wipe before placing in safety cabinet. 2) Pipette 10ML of prewarmed MEME medium into a 15ML Falcon tube. 3) Pour the stock cells into the medium as soon as they are defrosted. Swirl the contents of the Falcon tube a couple of times. 4) Spin for 5 minutes at 900RPM (must pellet the cells so that the old medium containing DMSO can be discarded, as DMSO is a growth inhibitor). 5) Use 25cm2 flasks for culturing and label on the top surface with the following information: name of cell line (e.g. Neuro2A), your name, date and passage number (e.g. PIII). 6) After spinning, carefully pipette off the medium using a 5ML pipette. It is important that the pipette does not make contact with anything other than the medium. The spent medium is disposed in a DispoJar filled with Virkon. 7) Gently pipette 5ML of fresh MEME medium into 15ML Falcon Tube containing cell pellet and slowly pipette up and down to resuspend the cells. 8) Transfer resuspended cells to a 25cm2 flask and spread around on its bottom surface. 9) Move the flask to the 37oC incubator and monitor their growth over the next few days. </p><p>1</p>