How many passages to wait after reviving before experiment - (Dec/10/2007 )

Dear fellow scientists,

After thawing cells, usually how many passages do you let the cell grow before using them for experiment for example transfection? I sometimes use the cells right away after they become confluent.

thanks.

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I would normally passage my cells at least once. Mostly because I need a larger number of cells for my experiments, but also to be sure that cells are fit after having been frozen. I don't think that there is a golden standard.

Kirsten
Aarhus University Hospital
Denmark

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definitly at least once - i'd go with two to remove any doubt

dom

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We usually go for two. You wouldn't want any DMSO in your experiments

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Thank you guys for your reply. I agree with all of you. I will wait at least for one passage before using the cells for experiments.

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you'll get better growth if you remove the dmso the day after thawing once the cells have attached - just replace the media - no passaging required

dom

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You could also dilute the thawn cells in fresh (cold) media, spin them for 10 min @ 800 rpm, remove the supernatant and then resuspend the pellet again in 10 ml media.

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You can use cells folloowing a thaw, not an issue. All you need to do is wash the cells 2-3 times, the DMSO comes off rather easily and quickly in my experience. You can passage the cells to just make sure they acclimate, proteins are expressed maximally and so forth but I would not worry. Many people use cells for assays right out of the liquid nitrogen. There are some functions, particularly for NK cells, that are blunted by the freeze thaw process. Nothing you can do will likely restore that function.

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I normally pass twice before doing anything, but once I did a quick transfection exeriment the day after I thawed CV-1 cells. It went well.

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Dear sumpf,

This is the correct answer but not to the original question. I totally agree with you about centrifuging at a low g (please do not use RPM as this does not mean anything...it has to be in g).

Oh dear, me and Dom clash again. In our studies we have found over the years that keeping cells in DMSO + media overnight is more damaging to the cells than centrifuging and reducing the DMSO concentration at Time 0.

The answer to the original question is.......it varies with different cell lines and primaries. As a general rule at least 1 or 2 passages are required. For J774 cells....the easiest and least destructable cells in the world....you do not have to even passage them for experimentation.

Kindest regards...even to Dom

Rhombus

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