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Splenocytes from C57BL/6 mice were stained with CD54 (ICAM-1) antibodies and analyzed by flow cytometry using the MACSQuant®
Analyzer. The specificity of the conjugated antibodies was confirmed by blocking the binding to the ligand, using pure unconjugated antibodies (left peak). Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of tandem conjugates.
In order to compare the epitope specificity of REAfinity Clone REA171 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 REA171|
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