Admin: Created page with "= Freezing Mammalian Cells = #In a T75 flask, grow cells to 80-90% confluency, or 75-80% density for suspended cells. #Collect cells (trypsinize/scrape adherent cells, etc)...."

2021-02-01T19:26:18Z

Created page with "= Freezing Mammalian Cells = #In a T75 flask, grow cells to 80-90% confluency, or 75-80% density for suspended cells. #Collect cells (trypsinize/scrape adherent cells, etc)...."

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= Freezing Mammalian Cells =

#In a T75 flask, grow cells to 80-90% confluency, or 75-80% density for suspended cells.
#Collect cells (trypsinize/scrape adherent cells, etc). Pellet using a 500g (1500RPM) spin, 5min at 4oC.
#Suspend cells in 4ml of culture media containing at least 10% FCS.
#Add 1ml of DMSO, mix well.
#Aliquot into labelled cryotubes, 0.5-1ml per tube.
#Place in a cell freezer that has equilibrated to room temperature
#*Mark the container (on tape, on the lid) to indicate a use
#*After the 5th use, the isopropyl alcohol must be replaced with fresh alcohol
#Put cell freezer in a -80oC freezer. This will cool the cells at ~1oC/min. Let sit in -80oC overnight.
#Transfer tubes to liquid nitrogen store
#Make sure to enter the cells into the cell storage database.

 

= Thawing Mammalian Cells =

#Warm 10ml of the appropriate tissue culture media to 37oC
#Remove a frozen tube from the liquid nitrogen store and transfer immediately to a 37oC waterbath
#Place 10ml of media into a T25 or T75 flask (T25 will achieve confluency faster). Pipette thawed cells into media
#Place in 37oC incubator. 12-18 hours later exchange the media in order to remove the DMSO

Freezing and Thawing Cells - Revision history

Admin: Created page with "= Freezing Mammalian Cells = #In a T75 flask, grow cells to 80-90% confluency, or 75-80% density for suspended cells. #Collect cells (trypsinize/scrape adherent cells, etc)...."