Hello,
I am seeding SH-SY5Y cells in 24 well plates, to be used for Zika plaque assay. Seeding cells, quite high, at 2.5 E6 overnight to achieve ~90-100% confluency, however cells are 'peeling' from the plate. I have tried pipetting gently down the side of the well and this helps a lot, although I still have problems with cells randomly peeling away.
I know I am seeding with a high concentration of cells, however when I seed with less I dont get high confluency, which I need for the plaque assay. I have been doing this with Vero cells (seeding at 1.5 E6) and have no problems.
Any help or tips, much appreciated.
Thanks,
Sarah
Hi Sarah,
This is a frustrating issue many of us face with certain cell lines. If you are not able to change the cell line to one that doesn't peel off, then there are ways to minimize peeling. Assuming you are being very careful not to cause much media movement back and forth which might disturb the sheets and that cells are peeling when you wash or change media, one approach I've found helpful in extreme circumstances is not to remove all the media from the well. Instead, one can remove most of it, and then add new solution slowly, then remove most of that solution from the well but not all, and repeat, essentially diluting the initial media enough so a complete media change has been accomplished. This, removing and adding media, is best done with a hand-held pipette rather than a vacuum for removal and a pipetteman for addition. Also, you may try using poly-lysine coated plates if your assay can accommodate them. Moreover, rather than trying to get complete confluence where cells are all connected to each other on the next day, if you optimize the cell number that is seeded, you will find a sweet spot where they are very near confluent and attached to the plate rather than to each other. This prevents sheet formation and therefore they will not peel off as one. Good luck!
Sarah, I has the same problem but now my protocol is working. I'm seeding 5x10^5 cells per well. Make sure your media is at room temperature (36~37C)...I've been leaving my DMEM/F12 for at least 20 min in the water bath and this made a HUGE difference.
Hi Sarah,
Try to coat your plates with gelatin, poly-L-lysine or other coating solution. After planting the cells, give them more time to attach well on the surface of the plate and than start the assay.
Best of luck!
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