Sean Lauber:Freezing Mammalian Cells: Difference between revisions
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(New page: I like to freeze down enough cells to comfortably expand them in a T25. Depending on the cell line this number could vary. I always keep an additional flask with cells to ensure I still ha...) |
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I like to freeze | When freezing cells I like to think about how many times I can split a flask into a T75 (to be ready for the next day) to give an idea of how many vials to freeze per flask. For instance, if I have a confluent T150, and I can split that T150 to make 4 T75s which would be ready for the next day (assuming a 1:2 split is appropriate for 24 hour confluency), then I would make 4 vials out of a single T150. It's a good idea to keep an additional flask with cells to ensure I still have cells even if something goes wrong with this process (and kills all my cells). | ||
#Prepare cells for collection (trypsinize, scrape, etc.) | #Prepare cells for collection (trypsinize, scrape, etc.) | ||
#Collect cells into an appropriate vessel (50 ml falcon tube) | #Collect cells into an appropriate vessel (50 ml falcon tube) | ||
#Spin at | #Spin at 1000 RPM/4*C/3 min to pellet the cells | ||
#Remove the supernatant and resuspend in appropriate media supplemented with 20% FBS/5% DMSO | #Remove the supernatant and resuspend in appropriate media supplemented with 20% FBS/5% DMSO. <b> At this point try to work quickly as the DMSO is killing your cells </b> | ||
#Remove an aliquot of cells for viability counting if desired | #Remove an aliquot of cells for viability counting if desired | ||
#Pipette 1 ml of the cell suspension into labeled cryovials | #Pipette 1 ml of the cell suspension into labeled cryovials | ||
#Store at -80*C overnight in a Mr. Freezie container (containing isopropanol) | #Store at -80*C overnight in a Mr. Freezie container (containing isopropanol) | ||
#The next day remove the cryovials to liquid nitrogen | #The next day remove the cryovials to liquid nitrogen |
Latest revision as of 21:36, 10 July 2013
When freezing cells I like to think about how many times I can split a flask into a T75 (to be ready for the next day) to give an idea of how many vials to freeze per flask. For instance, if I have a confluent T150, and I can split that T150 to make 4 T75s which would be ready for the next day (assuming a 1:2 split is appropriate for 24 hour confluency), then I would make 4 vials out of a single T150. It's a good idea to keep an additional flask with cells to ensure I still have cells even if something goes wrong with this process (and kills all my cells).
- Prepare cells for collection (trypsinize, scrape, etc.)
- Collect cells into an appropriate vessel (50 ml falcon tube)
- Spin at 1000 RPM/4*C/3 min to pellet the cells
- Remove the supernatant and resuspend in appropriate media supplemented with 20% FBS/5% DMSO. At this point try to work quickly as the DMSO is killing your cells
- Remove an aliquot of cells for viability counting if desired
- Pipette 1 ml of the cell suspension into labeled cryovials
- Store at -80*C overnight in a Mr. Freezie container (containing isopropanol)
- The next day remove the cryovials to liquid nitrogen