Hey all
Usually, at what confluency is a wound healing assay performed?
I have cells below 70-80% confluency in a 6-well plate.
Is this stage enough to do the assay?
Hello V H Krishna Prasad
No, cells below 70-80% confluency will not help for a wound healing assay.
The information derived from the wound healing assay is the rate of gap closure, which is a measure of the speed of the collective motion of cells. You will have to obtain a confluent monolayer of cells (nearly 100% confluency). The wound healing assay should be initiated at the same time point after the cells become confluent. It is important not to delay further because the results obtained may vary as the cell monolayer matures.
Best.
Thank you very much for your reply Malcolm Nobre
Could you also point out whether it will be okay for me to wait until the cells reach confluency, or should I start seeding in another six-well plate (10,000 cells/well).
Hi! For scratch assay it is required to have almost 100% confluence. In my experience, if the cells stay in the culture for a long time until they reach confluence, they no longer migrate the same way as when they are seeded for 24-48 h. It also depends on the cell line, of course. I suggest you to start a new experiment and seed the cells at a higher cell density. I think 24 h of incubation is best, but 48 h can work too. More than 48 h is too much.
V H Krishna Prasad, I agree with Andreea Iosageanu . Seed cells at a higher density such that they become nearly confluent after 24 hours. Will 10000 cells/well give you near to 100% confluency in 24 hours in a 6-well plate? If you are not sure, you may perform a pilot experiment with different seeding densities and decide on the optimal seeding density that gives you nearly 100% confluency in 24 hours.
Thank you very much, Andreea Iosageanu and Malcolm Nobre
I had done a pilot experiment previously with 10000 cells per well which gave me nearly 100% confluency. That ought to cover it. Thank you both again
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