Storing cells in liquid nitrogen

Freeze at least four vials from each line; when working with an unknown cell line, thaw a vial and check for cell viability one week after freezing. Freeze the cells at an early passage. Please note the passage number and other details to the data sheet in the reference filer.

freezing mammalian cells

  • grow cells in a 75 cm2 flask 7-75, at confluency cell density is 1.5x107 cells
  • wash cells with PBS
  • trypsinize with 3 ml
  • incubate for 5-10 min at 37¡C. Check the cells once in a while for detachment
  • resuspend cells in medium (10 ml)
  • pipet carefully to remove all cells from the flask
  • spin cells for 2 min at 200 xg (1000 rpm in Heraeus centrifuge)
  • aspirate supernatant
  • resuspend cells gently in 10% DMSO/20% FBS, use 2 ml
  • final concentration of cells should be 4x106 cells/ml
  • transfer twice 1.5 ml into 2 ml cryo-tubes (check that tubes have silicon sealing rings), thereby having two vials/cell line
  • place directly in -70¡C (storage for a couple of weeks, not longer)
  • 24 h later transfer to liquid nitrogen for long term storage

freezing Drosophila cells

  • grow cells in a 75 cm2 flask 7-75, at confluency cell density is 1.5x107 cells
  • spin cells for 4 min at 200 xg (1000 rpm in Heraeus centrifuge)
  • discard medium (supernatant)
  • resuspend cells in about 2 ml Schneider medium + 20% FBS + 10% DMSO to a final concentration of 5x106 cells/ml and freeze in 1 ml aliquots
  • place the tubes to -70¡C (storage for a couple of weeks, not longer)
  • 24 h later transfer to liquid nitrogen for long term storage

thawing:

  • remove cryo-tube from liquid nitrogen, use ice (short term) or dry ice for transport
  • prepare a 15 ml Falcon tube with 10 ml appropriate medium
  • use a 37¡C degree water bath for thawing but take care to keep the lid of the vial above the surface of the water to lessen the chances of contamination
  • when the cells are almost thawn (if only a little chunk of ice is remaining), wipe the outside of the vial with 70% ethanol and transfer cell with a 5 ml pipet
  • mix cells gently and spin for 2 min at 200 xg
  • remove the medium and resuspend cells in 10 ml and transfer into a 10cm2 dish
If cells are very sensitive like GH3, INS-1, etc wash for a further time after thawing. If cells have a poor viability after thawing than supplement the medium with 20% FCS and 1 mM sodium pyruvate)