Thawing 

  1. Pre-warm complete media. 
  2. Place vial of cells in a 37 ºC water bath for 2 minutes until nearly (~ 80%) thawed. Don't leave for too long because DMSO in freezing media makes cells sad. 
  3. Remove the cells from the vial and add slowly into a 15 mL conical tube containing 10 mL pre-warmed Complete Media (DMEM, 10%FBS + NEAA + PenStrepGlut).
  4. Centrifuge for 3 minutes at 1000 × g to pellet cells and remove the supernatant.
  5. Resuspend cells in 10 mL Complete Media, add to a culture dish and put cells in 37C incubator. 

Freezing

  1. To make freezing media, add DMSO to Complete Media (DMEM, 10%FBS + NEAA + PenStrepGlut) to 5% final concentration
  2. Count cells and make sure density is 1-5 x10^6 cells/mL. 
  3. Aliquot into cryotubes (the tubes with the orange caps) with 1mL in each tube. 
  4. Add isopropanol to the isopropanol tube holder things (see picture below), place cryotubes in the tube holders. 
  5. Place container in the -80, leave overnight.
  6. Remove tubes from the isopropanol container and place in -140.




  • No labels