Hi all,
I'm currently doing co-culture of MSCs and PBMCs in a 96 well plate. MSCs are left to adhere for 24 hours, before adding CD3/CD28 activated PBMCs (1:10 ratio). However, I find my supposedly non-adherrant PBMCs are getting stuck to the MSCs. Any ideas on how to prevent this?
It's important as my assay output needs to consider differences in MSC growth due to their cell culture media and the stickiness of PBMCs is messing with the results.
Thanks!
Hi Rebecca,
Before you add the pbmc, you should allow the cells to adhere to a polystyrene t flask for 2 hours at 37 to remove monocytes. Then use media to wash off non adherence cells and use that fraction for your assay
Hi Lora Benoit. Thanks for your answer - I probably should have mentioned, but I do rest PBMCs for 24 hours in a T25 before using them as I've heard this increases viability. I don't think the problem is purely monocytes - they all seem to stick close to the MSCs and its very hard to wash off.
Dear Rebecca,
You can use larger plate and an insert in the plate. The mscs are grown on the plate and the pbmcs are put on the insert. So they are separated by the membrane of the insert.
Good luck.
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