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Naive CD4 + T cells were isolated from human peripheral blood mononuclear cells (PBMCs). Cells were either left unstimulated (left images) or stimulated with PMA (50 ng/mL) and Ionomycin (1 µg/mL) for six hours. After two hours, brefeldin A (1 µg/mL) was added to the stimulated and to the unstimulated cells. Cells were fixed, permeabilized, and intracellularly stained with Anti-IL-17A antibodies as well as with CD3 antibodies. Cells were then analyzed by flow cytometry using the MACSQuant ® Analyzer. The Tandem Signal Enhancer has been used to increase binding specificity of tandem-dye–conjugated antibodies. Cell debris were excluded from the analysis based on scatter signals. |
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In order to compare the epitope specificity of REAfinity Clone REA1063 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 REA1063 |
| CZ8-23G1 | ++ |
| BL168 | ++ |
| N49-653 | ++ |
| eBio64DEC17 | - |
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