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Human peripheral blood mononuclear cells (PBMCs) were either left unstimulated (left peak) or stimulated with CD3/CD28 antibodies for 15 minutes on ice followed by stimulation with goat anti mouse IgG for 15 minutes on ice, and then allowed to undergo phosphorylation at 37 °C for 5 minutes. Cells were fixed and permeabilized using the Cell Signaling Buffer Set A followed by intracellular staining with Anti-LAT pY171 antibodies. Flow cytometry was performed with the MACSQuant®
cells were pregated for the analysis. Cell debris were excluded from the analysis based on scatter signals.
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