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now: Thu Jan 28 00:42:40 2021 ... mod: Mon Dec 21 20:28:22 2015
Ahhhh .I don't know if I'm brave enough for a frdgie photo! I feel like I've been busy/gone, that my entire place needs a deep clean. The frdgie being on the top of the list. Ha! That's a brilliant tip for the frdgie/freezer. I'll start there.
Thawing Tissue Culture Cells
Very easy. The goal is to get the frozen cells transitioned to growth temperature and conditions as rapidly as possible.
- remove the vial from the liquid nitrogen freezer
- in a laminar flow hood, wearing latex gloves, wipe down your gloves and the complete outer surface of the vial with a 70% ethanol solution (to minimize contamination)
- prepare a dish with medium of the same size the cells were initially collected upon for freezing (eg, if the cells were initially collected for freezing from a 10 cm diameter tissue culture plate, thaw the frozen cells back onto a new 10 cm diameter plate). put the dish with media in the incubator to equilibrate temperature and pH
- replating the cells after thawing at either too high or too low a density is not good
- unscrew the vial to equilibrate the gas pressure in the vial with normal atmospheric pressure, then screw the lid back down to seal the vial
- hold the vial in your (latex) gloved hand until it is partially thawed. alternatively: incubate with vigorous agitation in a 37C water bath until completely thawed
- this won't hurt your hand -- the vial doesn't have that much heat capacity in it and will very rapidly warm up once removed from the freezer
- shake the vial until the remaining frozen material in the vial sloshes back and forth against the melted solution in the vial
- remove the cap of the vial and dump the half-frozen cell slurry directly into the medium in the previously prepared plate
- swirl the plate until the remaining frozen material has completely thawed, then immediately place the plate in a humidified tissue culture incubator
see also: FreezingTissueCultureCells
- in general, do NOT add selective drugs to the medium until 24 hours after thawing