Hi
i electroporated U937 cells and i want to store them for further investigations.
What are the optimum conditions to do so?
Many thanks
Hello John Abdo
You can store the electroporated U937 cells at -80 degree C in an appropriate freezing medium containing cryoprotectant like DMSO after having given sufficient time for the cells to recover.
Best Wishes.
General freezing protocol for mammalian cells:
1) Trypsinise a healthy culture. Pipette up and down gently to separate clumps; ideally pass through a cell strainer to ensure single cell suspension.
2) Pellet cells by gentle centrifugation.
3) Resuspend pellet in freezing medium (6:3:1 DMEM:FBS:DMSO, no antibiotics). For one 90% confluent T75 flask I use 10mL freezing medium.
4) Aliquot 0.5-1mL into freezing vials (o-ring, internally threaded). Do not use Eppendorf tubes for this, they are prone to contamination or bursting (safety hazard). Close tubes tightly.
5) SLOWLY reduce the temperature - aim for a controlled cooling of 1 degree C per minute. Ideally, use a cell freezing device. If not available, insert tubes into a plastic float (normally used for supporting tubes in a water bath) and float them in a significant volume (300-500mL) of room temperature isopropanol inside a plastic container, then put this into a -80C freezer. Alternatively, as a last resort, wrap tubes in tissues and put in the fridge for several hours, then the -30 freezer overnight, then the -80 freezer overnight (this will reduce viability). DO NOT put open containers of isopropanol into a domestic freezer (flammable vapour is a fire hazard).
6) Frozen tubes can be stored at -80, but are better stored in liquid nitrogen vapor phase. Tubes that are at -80 can be transferred into LN2 immediately without any control of the freezing rate.
To recover frozen cells:
1) Quick thaw in the water bath.
2) Mix entire contents of vial with 10mL of pre-warmed culture medium.
3) Pellet cells by gentle centrifugation and discard supernatant. (This is important: DMSO is toxic to growing cells and must be removed.)
4) Resuspend in pre-warmed culture medium (use the volume appropriate for the flask/plate the cells will be grown in)
5) Transfer cell suspension to plate/flask and incubate. Check cells daily, change media frequently to remove dead cells.
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