Dear All ,
we are working on gene transfection by Calcium Phosphate method but after 24h when I want to change the cell culture medium of my test and contro cells , I see that tests are not as normal as controls as you can see in picture.
Are my cells dead? Why?
what is the problem ?
Any comment or suggestion would be highly appreciated.
Best
Niloofar
I'm guessing that the image showing low confluence is the one taken after 24 hours and not the other way around. In that case I think the problem is toxicity.
Toxicity may be caused by the transfection reagent or the protein you are trying to express. If the protein is not toxic I would suggest replacing the medium sooner (4-6 hours after transfection) or using a different transfection reagent (PEI or PEI-based).
Goodluck.
Dear @Omershkedi,
Thanks for your answer.
Actually I'm trying to set up this method in order to lentivirus preparation.but in this stage, I just transfect pCDH-GFP to recognize the efficacy of the method.there is no protein expression yet.
We have a protocol for lentivirus production in which we use PEI for transfection. This is a home-made transfection reagent that is cheap, easy to make and usually doesn't cause toxicity. I can share the protocol here if you'd like to try. By the way, what cells do you use?
Yes ofcourse. Thank you
HEK293t is our packaging cell line and CHO-K1 as host cell.
Hi, I am frequently doing transfection to PlatE cells (derived from HEK293t) using calcium phosphate method for retrovirus production. Firstly, calcium phosphate complex with DNA is toxic to the cells when left for too long. I generally transfect in the evening and change medium next morning. Changing medium ~1 hr before transfection and supplementing the medium with 25 uM Chloroquine enhances transfection efficiency quite well. However, chloroquine is also toxic, so changing medium within 16 hrs after transfection is necessary for better cell condition. Amount of DNA used may also affect cell condition, what amount of DNA do you use for transfection? As mentioned by Omer, PEI is easier to handle, you can try it as well. However, for me calcium phosphate generated higher virus titers compared to PEI.
Dear @nazimmedzehidov,
Thank you so much for your answer.
1-How much chloroquine should I use for each well in a 6-well plate?
2- according to the kit protocol, 1-3ug plasmid is needed for each well which is plated with approximately 3×10^5 cells
Dear Niloufar,
1) I generally use 25 µM chloroquine final concentration and it works very well.
2) Amount of DNA seems quite okay, so you might consider not letting your cells to be exposed too long to the DNA precipitate and chloroquine. Actually around 4-6 hours is quite sufficient for transfection to happen. But still I prefer doing it overnight.
Hope it works for you!
Nazim
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