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Human peripheral blood mononuclear cells (PBMCs) were either left unstimulated (left images) or stimulated with CytoStim at 37 °C for 16 hours. Cells were then stained with CD134 (OX40) antibodies as well as with CD4 antibodies. Flow cytometry was performed using the MACSQuant®
Analyzer. The Tandem Signal Enhancer has been used to increase binding specificity of tandem-dye–conjugated antibodies. 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.