I transfected my cells with plasmid DNA and tried to perform matrigel invasion assay. The problem is there is about %60 reduction in cell viability in both my pDNA and mock vector transfected cell when compared to control group if I perform overnight serum starvation. I treated the cells transfection reagent (TR) without plasmid DNA and validated that TR dramatically reduces a cell viability so I reduced transfection reagent as much as I could but the result is the same. In the link given below, it is stated that overnight serum starvation is not necessary; I can resuspend the cells in serum free media before starting the assay.
https://www.cellbiolabs.com/faq/cell-based-assays-faq/cell-invasion
I am really confused because our common protocol is to starve cells in serum free media overnight and perform the assay by adding %10 serum in lower chamber to create a chemoattractant gradient while performing the assay.
This situation really limits me because besides invasion assay; I can not perform scratch assay either. I can clearly see the space between cells in TR treated cells and once I create the wound, the cells scatter.
At this point, using a different transfection reagent is not an option for me.
I am open to any suggestions, thanks already.
Simple try not starving the cells as the vendor suggests, or reduce serum to 5% or lower just enough to allow cells to proliferate post TR. Do a six well plate two for 10% serum (full medium) two for 5% serum, and 2 for serum free compare and contrast viability as it could be a TR sensitivity persistence in your cells rather than serum requirement. It is best to start with a viability count and make sure all wells take roughly the same amount of cells. Reducing transfection time will reduce number of transfected cells. Can you not wait 24 hrs post TR then on the third day do your invasion assay when the cells have recovered in full medium and starve them to do invasion assay on 4th day? The info you provided is limited so my suggestions are based on what you stated. Good luck.
Try to reduce the transfection time and either add serum after 1,5 to 3 hours post adding the transfection reagent and let the cells recover o/n.
Hello Murat
Let me first know from you certain things. Are your cells adapted to serum free medium? If you are growing cells in 10 % serum and then subject them to serum free medium they are definitely going to undergo shock as a result the viability is going to decrease. I agree with Rabeah. You check the viability in 10 %, 5% and serum free. If you find 5% giving a good viability then try another experiment with 5%, 3%and 1%. The lowest serum concentration that gives good viability can be used instead of starving the cells overnight. The issue of TR can be dealt with at the next step.
Thank you.
Rabeah Al-Temaimi thanks for the reply. Well; first of all I like to mention that I wait about 12 hrs on the third day to give time the transfected cells to recover from transfection and then starve them overnight to do invasion on the 4th day. As i see under the microscope, there is nothing wrong morphologically after the transfection (i mean on the 3rd day) when compared the control group. The confluence is also similar when compared the control cells. It is a pity that i see a 50-60% reduction in viability after overnight starvation. I tried to reduce serum to 5% instead of starving cells and the result is the same. To be sure that the problem is TR related, I treated the cells with TR (without any plasmid) and the result is the same :/. What's more, I reduced the transfection time and TR amount (3 time lower than the manufacturer advised-validated the transfection was successful with PCR) but still an unacceptable reduction in cell viability. I have used this brand of TR on other cell lines and did not see this kind of problem. May be this cells are not suitable for this brand of TR. My budget is limited so I can not change the TR.
Malcolm Nobre
Hello Malcolm, thanks for your interest. Yes I a growing the cells in 10 % serum and then subject them to serum free medium. I performed a pre-experiment to see if serum starvation for overnight shocks my cells. So I compared the viability between 10% and 0% serum treated cells and did not see any reduction in viability.
To be sure that the problem is TR related, I treated the cells with TR (without any plasmid) and saw that the viability in TR given cells dramatically reduced compared to control group. So, I tired reduced the transfection time (9 hours instead of 24hrs) and TR amount (3 time lower than the manufacturer advised-validated the transfection was successful with PCR) but still there was an unacceptable reduction in cell viability. I concluded that this is a transfection reagent problem. One more thing, I also perform siRNA transfection with the same cell line using a different TR for siRNA transfection and haven't seen a dramatic reduction in cell viability (5% may be 10% at most). I am pretty sure that my problem is TR related. Yesterday instead of overnight starvation, I starved the cells prior to invasion assay and lowered the assay time for 9hrs instead of 24hrs to prevent reduction in cell viability and the results are pretty satisfying. I will let you know for any update and open to any further advise. Thanks again.
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