Murine fibroblasts can be tough to transfect, but with appropriate reagents and protocols (https://altogen.com/product/mef-transfection-reagent-mouse-fibroblast-cells/) you can get much higher efficiencies. In lipofection-based approaches, serum-free medium isn't really a necessity, so you can run the transfection without fearing for cell shock to changed environments. If your cells are getting too confluent, try starting with a lower confluency (by passaging) before transfection, and that should keep them from crowding too much.

I don't have any idea about GSK3B -/- cells. But I used do transfect MEFs very frequently. I think you could split your cells and harvest those cells at 11:30 pm. But remember one thing, after splitting the cells will be in medium without transfection reagents/DNA. Would that work good for your experimental condition? If it does not work, then leave the cells as usual without doing anything and harvest them in time. I think over-confluence would not matter too much.

Hi

ARUN THANK YOU for the suggestion. I think this one works in my case, I will split and harvest until my desired time point to collect the cell pellet.

Once again thank you so much for early response

ramesh

It is normal for the cells to grow after transfection. The only way to stop would be to add the selection agent which is not advisable within the 24 hrs of transfection. I believe you may have transfected when your cells were about 80% confluent, and 36 hrs after that would definitely lead to fully confluence. You did not mention why you needed the cell pellet, if your purpose was to check any gene expression then I would not suggest subculturing them without selection since further culturing would increase the non transfected cell population, and the cell pellet at 36 hrs should be good enough for westerns. Hope this helps.

Does what you transfected into the cells promote growth? Do you see the same confluency in vector-transfected or non-transfected cells? Is this a result of the gene you transfected in? Things to think about.

Hi Chandra,

I need the cell pellet for protein expression and your suggestion worked well. Usually these MEF cells grow very fast so usually I do when it is 80% confluent but here I m afraid before 36 hrs only it may over grows but itcouldnot happened. Thank u once again

Hi Richard,

After 6 hrs of transfection I usually change the serum free media to the serum media.i see not much confluency in vector transfected cells. Need to have some more observations to discuss about this.

Murine fibroblasts can be tough to transfect, but with appropriate reagents and protocols (https://altogen.com/product/mef-transfection-reagent-mouse-fibroblast-cells/) you can get much higher efficiencies. In lipofection-based approaches, serum-free medium isn't really a necessity, so you can run the transfection without fearing for cell shock to changed environments. If your cells are getting too confluent, try starting with a lower confluency (by passaging) before transfection, and that should keep them from crowding too much.