Abstract
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 different cell signaling pathways. These pathways include T and B cell signaling, regulating growth and cell cycle control, plus cytokine, chemokine, and stress responses. Phosphoflow assays combine phosphoprotein-specific antibodies with the power of flow cytometry to enhance phosphoprotein study. In our assay, peripheral blood mononuclear cells are stimulated by cytokines, fixed, surface-stained with a cocktail of antibodies labeled with MAXPAR (brand name) metal-chelating polymers and permeabilized with methanol. They are then stained with intracellular phospho-specific antibodies. We use a CyTOFTM mass cytometer to acquire the ICP-MS (inductively coupled plasma mass spectrometry) data. The current mass window selected is approximately AW 103-203, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type. Median values are reported to quantitate the level of phosphorylation of each protein in response to stimulation. Comparing the level of phosphorylation between samples can offer insight to the status of the immune system.
Materials and Reagents
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Representative data
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Acknowledgments
This work was supported by grants S10RR027582, 5U19AI057229, and 5U19AI090019 from the U.S. National Institutes of Health.
References
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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.
When using soluble CD3, I stimulate for 15 minutes and fix. CD3 = 2.5 ul in 990ul ( Final conc 500ng/ml) BD Pharmingin Catalog # 555329CD28 = 10 ul in above media Final ( conc 2000ng / ml) BD Pharmingin Catalog #555725I get very good stimulation of pP38, pPLCg2,pERK1/2, pS6, pSTAT3, IkB, pSTAT5 and moderate stimulation of pAKT and pSTAT1
thanks, very helpful!