Helo dear colleagues
After transfection and a 48h drug treament we want to assess the motility of our Glioblastoma cell lines.
Is it important to use a ECM coating to perform this assay? (during transfection/drug treatment there was no ECM protein coating)
Our timepoints will be 6 and 18 hours. The doubling time of the cells is ~24h.
Do you expect major implications of the proliferation in the assay?
Should we perform an over night serum free incubation or perform the experimente without serum? (cells can handle serum free media)
Thank you
As you mention, the cellular migration potential should be evaluated using the ECM-coated dishes. However, serum depletion does not necessarily be required.
Thank you very much.
Would poly-L-lysine or ornithine be a good choice?
And with the scratch I will damage the monolayers, so cells will possibly migrate towards a space lacking this ECM protein. Isn't that a problem?
Hi Pedro,
as per my experience with many GBM cell lines, you don't need to add any ECM for Scratch assay. your time point might need to change as since your cell line doubling time is 24 hrs. I would recommend to take picture at T=0 (usually its T=1 hr for smooth surface around scratch) and then T=24 and 48 hrs. If controls won't get complete wound closure, you can extend the time to T=72 hr for clear phenotype.
I wouldn't go for poly-lysine it will certainly hamper the migration.
More info, you can look at our labs article in this matter.
http://www.ncbi.nlm.nih.gov/pubmed/25275602
http://www.ncbi.nlm.nih.gov/pubmed/25592037
All the best :)
Thank you very much for the aswers Dr. Yoshida and Dr. Tomar
I have now all the information needed to proceed with the experiment.
Cheers
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