I perform scratch assay by over-expressing or adding drugs, like doxorubicin or hydroxyurea. I looked for various protocols online, but there is no clear procedure as to how long the transfection should be done before the scratch. I have tried by doing the transfection for 4 hours followed by scratching (I do not change the media after scratching although I wash the floating and hanging cells). I later take images until 24hrs after scratch. I use HeLa and HCT116 cell lines.
I really appreciate valuable inputs. Thank you.
Supraja
Hi Supraja, it depends on the cell context and cell types. Generally speaking, if you want to see the effect of an over-expressed gene on cell migration, you have to wait until the target gene is indeed overexpressed. Therefore you need to figure out the best time point. If I were you, I'll firstly measure the levels of target protein 24-48 hrs after tranfection; if the protein is indeed overexpressed, I'll start scratch assay from then. If you want to see the effect of a centain drug on cell migration, you can start scratch assay along with drug addtion.
I would like to express a long non-coding RNA and perform scratch assay. I observe that after 24 hours of transfection with the lncRNA, most of my cells are dead and floating. Could anyone suggest a detailed protocol as to how long I should wait, after transfection, before performing scratch assay?
Dear Supraja,
in order to obtain a helpful response from the RG community you need to describe your problem as concise and short as possible. Obviously you intend to do scratch assays with transfected cells. Without further details on what you are going to transfect most people would assume that it is an expression vector for a protein. Hence Han Zhang gave you excellent advice. A scratch assay works on a time scale of many hours to days. If your transfection kills your cells within 24 hours it does not make sense to perform such an assay unless you find out the reason why your cells are dying. Science means to perform the proper controls, but only you know what the proper controls are. Is the transfection procedure killing you cells? Did you transfect a control RNA? What controls did you do?
You are looking for a clear procedure, but that is not how science works. You have to figure out the procedure that is working for you! You have to become a scientist!
Please be aware that there have been a few discussions on the scratch assay on RG in the past.
Good luck with your experiments.
Thank you for your response Dr.Klug. After trying out various time points and different methods for performing the assay, I figured out a protocol that works best for the conditions I am looking for. I obtained great results with the protocol I have optimised. Thank you for your suggestion.
Supraja
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