Rosemary Fernandez —BIO-PROTOCOL

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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

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

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: 10893, Q&A: 1

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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Neha Rana

University of Bergen

Hi,
Can this protocol be worked out on whole blood samples directly fixed with protein stabilizer and stored at -80C ?

Thank you.
Neha

5/21/2019 2:30:35 AM Reply

Holden Maecker

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

No, that’s not possible once the cells are in Proteomics Stabilizer, as it contains a fixative.

2019-05-24 07:23:10

Neha Rana

University of Bergen

Thank you very much for the reply . Yes , I understand that , But I actually meant if cytokinins stimulation be done in freezed samples . That doesn’t looks like a possibility though !

2019-05-23 23:28:08

Holden Maecker

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

Yes, it's absolutely fine to skip the resting/stimulation, put blood directly in Proteomic Stabilizer, and proceed from there. This allows for ex vivo phenotyping + assessment of basal signaling states.

2019-05-23 17:12:44