Rosemary Fernandez —BIO-PROTOCOL

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Coauthors

Holden Maecker Microbiology & Immunology, Stanford University, Stanford
7 protocols

Rosemary Fernandez

Human Immune Monitoring Center, Stanford University, USA

2 Author merit
Reviewer merit
Editor merit

2 Protocols published

Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry

Authors: Rosemary Fernandez and Holden Maecker, date: 06/05/2015, view: 7168, Q&A: 1

Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many ...

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Yvonne Vercoulen

UMCU

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 stimulation, and using P-ERK, P-S6 or P-Zap70 as readout. I noticed that you see some nice signaling profiles for cytokine stimulation. Does aCD3 or CD28 stimulation work for you on total PBMC? If so, can you please recommend a method to us?
Thank you & best wishes,
Yvonne Vercoulen

9/7/2016 5:44:08 AM Reply

Yvonne Vercoulen

UMCU

thanks, very helpful!

2016-10-31 23:08:13

Rosemary Fernandez

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

2016-10-31 15:08:44

Holden Maecker

Microbiology & Immunology, 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.

2016-10-31 08:42:07

Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry

Authors: Rosemary Fernandez and Holden Maecker, date: 06/05/2015, view: 5865, Q&A: 0

The ability to assess the function of a range of cytokine, antigen receptor, and Toll-like receptor (TLR) signaling pathways in a range of immune cells could provide a kind of fingerprint of the state of the human immune system. The mass cytometry ...

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