Prepare media ahead of time and warm to room temperature
Spray down each bottle with 70% Ethanol before placing it into the TC hood
Pre-cool the centrifuge to 4C
Prepare and label a 6-well plate, and aliquot 9mL of media into a 15mL conical for each tube of cells to be thawed
Find the location of the cells you will be thawing in the -80C freezer inventory google doc
Transfer the cells between the freezer and the TC room on ice
Record the information on the cryotube – date, number of cell, etc
Begin adding media from the 15mL conical to the still frozen cells, then transfer media back and forth between the two tubes until the cells have completely thawed and everything has been transferred to the 15ml conical
Spin down the cells at 1200rpm for 10’ at 4C
Pour off the supernatants and break up the cell pellets by tapping the tubes
Add 1mL of media per 2x10^6 cells to the cells, pipette up and down to break up any clumps, and transfer to the 6-well plate
Add 10ul of Mycoplasm removal agent per 1mL of media
Confirm the presence of cells in the well by microscope
Incubate at 37C