Hi everyone.
I am using Invitrogen CTS CD3/CD28 Dynabeads to simultaneously activate and isolate T cells from cryopreserved PBMCs. I thawed PBMCs in AIM-V medium, viability was 80% by trypan blue staining. During incubation with Dynabeads (RT, 1hr, with rocking) a big clump formed which did not dissociate upon pipetting. I then proceeded to removal of unbound cells and cultured the bound (presumably CD3+CD28+ T cells) cells in IL-2 supplemented medium. The big clump remained when I checked my cells the next day. Recovery of live cells was pretty poor and I suspect most of the cells either died/got trapped within the big clump.
Is this normal for cyropreserved PBMCs?
Hi Rachel Tam,
I think its the DNA from dead cell which is the cause for clump formation. Check protocol for reviving the cryopreserved cells. You can also add some DNase to the culture.
Agree, my instinct in your scenario is you had a big clump of dead cells. Check with trypan blue after thawing as well to answer that question.
Thanks for your reply.
Immediately after revival trypan blue stained cells were 20% but I do know that trypan blue cannot mark dying cells. I thawed my cells at 37C and washed in cold medium. Should I use pre-warmed medium instead to boost longer term survival?
Hi,
Please find attached nature protocol that will be answer to your question. in your case you need to use DNase during stimulation to avoid clump formation.
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