Hello, I isolate PMBCs from blood using Ficoll and plate the cells in a 96-well plate for different treatments for a few hours before T cell activation. At the end of the treatments I centrifuge the plate, discard media and use new media to pipette the cells several times and transfer to an anti CD3 coated 96 well plate for 3 days, new media is also supplemented with anti CD28 ab and IL2.
I can't seem to get the cells into the new plate and rarely get any cells in the plate after the 3 day incubation. I was under the impression that these cells detach easily with pipetting. Any suggestions?
You should may be try Corning® Costar® Ultra-Low Attachment plates. I'm using them for PBMC derived macrophages (in 6-well plates others formats available) and they worked great for me.
As far as I know, lymphocytes shouldn't attach at all. My suggestion is to look at the plate that you centrifuged after "transferring" the cells to the new plate. My thinking is you are either killing the cells by centrifuging at too high of a speed, or you are not mixing them well enough once you resuspend. Are you looking at the cells before centrifugation? They may be already dead for some reason.
Kerry S Vistisen Thank you for your reply. I am looking at the cells before and after centrifugation, they look healthy but I am not very experienced with them so I might be wrong. What conditions would you recommend for centrifuge?
I would keep the centrifuge around 170 g +/-. On our centrifuge, that is around 900rpm. But if they still look good after, then they should be okay.
You might want to look at the plate to make sure the cells haven't been left behind after you transfer them.
Kerry S Vistisen I tried to lower the g and doubled the amount I seeded, and it now looks like I was able to transfer a good amount. Thanks!
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