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Whole blood from a healthy donor was stained with the 7-Color Immunophenotyping Kit, human. Staining was carried out at 4 °C for 10 minutes. Subsequently, red blood cells were lysed by incubation using 1× Red Blood Cell Lysis Solution at room temperature for 15 minutes and analyzed by flow cytometry using the Express Mode of the MACSQuant®
As a preliminary step for elimination of doublets a gate around single cells in forward scatter area (FSC-A) versus forward scatter height (FSC-H) was set (A). To identify the major circulating blood cell types CD45 was used to target all leukocytes (B). Theses cells were further separated from debris via forward scatter (FSC) and side scatter (SSC) (C). CD19 has been used to define B cells and CD3 for T cells (D). The T cells were separated in CD4+
T cells (E). CD14/SSC intermediate were selected to identify monocytes (F). Monocytes were excluded and the remaining cells were further characterized. A gate was defined on CD16/SSChigh
cells to identify mature neutrophils, which could be separated from eosinophils due to the absence of CD16 expression. A CD16/CD56/SSClow
gate was used for sub-characterization (G). These cells were further characterized by gating on CD3/CD56+
T cells and CD56/CD16+
NK cells (H).
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