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monocytes were isolated with CD14 MicroBeads, human, from peripheral blood mononuclear cells (PBMCs) and cultured in the presence of GM-CSF and interleukin 4 (IL-4) for 6 days into inmature monocyte-derived dendritic cells (Mo-DCs). For generation of mature Mo-DCs, cells were cultured for another 24 hours in the presence of TNF-α, IL-6, and IL-1β. Mo-DCs were then stained with CD80 antibodies or with the corresopnding isotype control antibodies (left peak) and 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 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.
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