Product: Brilliant Violet 421™ anti-ERK1/2 Phospho (Thr202/Tyr204)

Catalog No.: 369509

Dejan Mesner, Mr UCL

Good Staining of Phospho-ERK in CD4 T Cells
Overall:	Product Quality:	Ease of Use:
Decent detection of ERK phosphorylation upon PMA/ionomycin stimulation of CD4 T cells.

Experimental Design

Application:	Flow cytometry
Cells used:	PBMC
Brief Protocol:	PBMC were isolated from buffy coat using Ficoll separation. CD4+ T cells were isolated using negative selection beads (Mojo sort kit). Cells were cultured (2.5x10^5 cells/well) in 20 IU/ml IL-2 overnight. Stimulation: 50 ng/ml PMA and 1 ug/ml ionomycin for 20min at 37°C. Fix: 4% formaldehyde buffer (15 min, 37°C). Wash. Perm: 200 ul ice cold 90% methanol (30 min, 4°C). Spin. Wash twice. Stain: 50 ul/well in PBS. pERK Ab used at 1:100. Wash Acquire (BD LSRII).
Results Summary:	We can observe increase in ERK phosphorylation upon PMA stimulation of resting CD4 T cells.
Additional Notes:	Lymphocytes were gated on FCS vs SSC plot. Doublets were excluded on FSC -H vs -W plot. Dead cells were excluded as Zombie UV+.

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