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Human peripheral blood mononuclear cells (PBMCs) were either left unstimulated (left images) or stimulated with 20 ng/mL PMA and 1 µg/mL ionomycin for 6 hours and after 2 hours with 1 µg/mL brefeldin A for 4 hours. Cells were then fixed, permeabilized, and stained with Anti-TNF-α antibodies as well as with CD69 antibodies. Flow cytometry was performed using the MACSQuant®
cells were pre-gated for the analysis. Cell debris were excluded from the analysis based on scatter signals.
In order to compare the epitope specificity of REAfinity Clone REA656 with other known clones, a competition assay was performed.
Cells were incubated with an excess of purified unconjugated REAfinity Antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker. Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.
|Other clones||Overlap in epitope recognition with REA656|