Hello everyone,
I am trying to starve LCL cells to study some autophagy related pathways, but when I starve the cells with EBSS medium without serum, the suspension LCLs become adherent right after 5 min or so. And after 4h of starvation nearly 90% of the cells are attached to the culture flask surface. As a result, I can not collect them as usual and I need to use trypsine. Even with trypsin it takes at least 15 min to detach them. And if I want to resuspend the pelletes after using tyrpsin, I have problems in dissociating them (they become dissociated only when I pipe them up and down with strong force).
Do you have the same problems? Looking forward to having some valuable suggestions.
Thank you all in advance
If you are using these cells for western analysis, you can grow then in 24 well plates and use the lysis buffer or hot loading dye directly on to the cells and collect the lysate. And if it is the flask just use a cell scraper and collect the contents and centrifuge. I use this strategy on the adherent cells normally. I hope it helps. Good luck.
To study autophagy, one can starve cells for increasing length of time (15, 30 and 45 min). Most cell types exhibit autophagy in 15-20 min. To detach adherent cells, one could use a cell scraper after mild trypsinization to maintain the cell viability.
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