rat1 cells freezing and thawing - how to culture rat1 fibroblasts (Dec/18/2006 )

hi all,
i need your guidance. i am trying to grow Rat1 fibroblasts after thawing them and they do not attach well to the plate even after 20 hrs in DMEM medium with 10% FBS, 1% P/S.

for thawing the cells i did rapid thawing at 37 degree water bath, followed by spinning down cells at 1.8 rpm for 5 min and plating cells in DMEM.

what could be the problem? they are not attaching well and look unhealthy.

i need your help urgently.

thanks
syc06

You said you use 1.8 rpm to spin down the cells, I assume your are using 1800 rpm. This speed is too high for primary culture, could you try 1000 rpm for 5 min.

Or don't spin down your cells, just dlute them in complete medium so that final DMSO concentration is less than 1% and seed them directly (without spinning) in the flask. Change the medium after cells are attached to the substratum.

Hope this helps.