Hi, for phospho-flow analysis we are trying to induce clear...

protocol Protocol: Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry
Hi, for phospho-flow analysis we are trying to induce clear T cell receptor signaling in total isolated PBMC (thawed). In Jurkat and other cell lines, as well as mouse T cells we can do it, but in total human PBMC we are not successful when utilizing aCD3/CD28 beads, or aCD3 antibody stimu Read more Read less more less
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Yvonne Vercoulen Answered Oct 31, 2016

UMCU

thanks, very helpful!
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Rosemary Fernandez Author Answered Oct 31, 2016

Human Immune Monitoring Center, Stanford University, USA

When using soluble CD3, I stimulate for 15 minutes and fix.
CD3 = 2.5 ul in 990ul ( Final conc 500ng/ml) BD Pharmingin Catalog # 555329
CD28 = 10 ul in above media Final ( conc 2000ng / ml) BD Pharmingin Catalog #555725
I get very good stimulation of pP38, pPLCg2,pERK1/2, pS6, pSTAT3, IkB, pSTAT5 and
moderate stimulation of pAKT and pSTAT1
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Holden T. Maecker Author Answered Oct 31, 2016

Immunity, Transplantation & Infection, School of Medicine, Stanford University, Stanford

Yes, we use CD3+CD28, either beads or soluble Ab, to stimulate PBMC for phospho-flow. For CD3+CD28 Dynabeads:
1. Exchange the supernatant of the Dynabeads with an equal volume of complete medium, by pelleting the beads with a magnetic separator.
2. Add 50 uL of Dynabeads in medium to 10^6 thawed, rested PBMC in a 96-well plate.
3. Stimulate for 30 min, then fix and proceed as per the published protocol.
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