Stable transfection of H295R cells - (Jul/30/2007 )
I am new to cell culture technique.I will be doing stable transfection of H295R cells, protocol suggest that after transfection transfer cells on 10cm culture plates from 12 well plate. But i don't know how to transfer transfected cells from 12 well plate to 10cm plate. please helpme ,any suggestion or procedure.
thanks in advance
Wash it with PBS without Ca Mg,
add 200 ul trypsin/EDTA,
add 200 ul more trypsin/EDTA,
incubate it rt untill cells get loose.
add 0.5 ml of culture medium with serum, pipet it to make a good suspension,
collect all the cells,
add the cell suspension to a 10 CM plate with 10 ml medium contianing selective agent.
wait till colonies to show up.
This is in addition to genehunter's suggestions.
After trypsinising the cells, you have to decide if you would like to plate 20 or 50ul from the cell suspension into the 10cm plate so that you get single colonies. It depends on your transfection efficiency. Too high efficiency, low volume of cells is sufficient.
even you get high transfection efficiency, but the integration is low. so after selection, a lot of cells will be died. I do not recommand transferring cells to 10cm dishes after transfection. what i recommand to do is doing transfection and selection in 6 well plate. then dilute cells survived and select clone in 96 well plate for establishing sigle cell clone