I want to determine glucose uptake in epithelial cells. I trypsinize the cells from the a stock flask and seed out a desired amount of cells in 96 well plates to perform the assay in. I am unsure about how long it would take for glucose transporter proteins to recover and be fully functional following trypsinization. I have not found any specific references in literature that answer this question. Any thoughts?
Since recovery is generally 6 hours.
I think the best option is to wait overnite after seeding at 5- 6 pm
Try to read this. it might be helpful.
http://www.ncbi.nlm.nih.gov/pubmed/8276890
Generally speaking, about 0.5x the doubling time suffices for repair of damages that may have occurred during stripping cells from the plates/flasks and seeding new flasks. Given that, 6hr or overnight waiting period should serve quite well.
I do recall Thorens paper on Glut2. Some of the conclusions that are relevant to isolated islets and Glut2 (beta cell glucose transporter) may not be applicable to other cells for several reasons, including the difference in size of the first extracellular loop on Glut2 versus other transporters, such as Glut1, which contains a much smaller extracellular loop between the transmembrane region 1 and 2 and has significantly different sequence. I have not tested it myself but I wouldn't be surprised if Glut1 turns out to be quite different as a substrate for trypsin.
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