1. Remove the sample from the freezer, and as it begins to melt, carefully aspirate the overlying oil from the cell/medium mixture.
2. Using a multichannel pipetter and working one row at a time, quickly, but gently multipipet the cells twice in order to resuspend them thoroughly.
3. Change the pipet to a P200 (set to 200 | L), and then quickly transfer the well contents one at a time to the individual wells of a 24-well plate. Pipet quickly to resuspend the cells in the 96-well plate. Then transfer. Once the sample is transferred to the 24-well plate, pipet again to resuspend the cells and distribute them evenly in the prewarmed fresh media.
4. Aliquot another 200 | L into the now empty well of the 96-well plate to rinse it, and remove any remaining cells.
5. Transfer this additional 200 |L to the equivalent well of the 24-well plate.
6. Repeat the above for each required well of the 96-well plate.
7. Place the 24-well plate containing the newly transferred cells in a humidified CO2 incubator at 37°C.
8. Change the media after 8 h or the following morning (if defrosting is carried out late in the evening), and then daily until the cells are ready to passage.
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