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Whole blood from a healthy donor was stained with the 8-Color Immunophenotyping Kit, human. Staining was carried out at room temperature for 10 minutes. Subsequently, red blood cells were lysed by incubation using 1× Red Blood Cell Lysis Solution at room temperature for 15 minutes. Cells were analyzed by flow cytometry using the MACSQuant ® Analyzer 10. 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) (A) as well as a gate around viable cells was set (B). To identify the major circulating blood cell types CD45 was used to target all leukocytes (C). Theses cells were further separated from debris via forward scatter (FSC) and side scatter (SSC) (D). Monocytes were discriminated based on their CD14 expression (E) and then further divided into classical, intermediate, and non-classical monocytes via CD16 (F). Among the non-monocyte population, B cells were defined as CD19 + (G). The remaining cells were separated into CD16 + /SSC high neutrophils, CD16 – /SSC high eosinophils as well as a CD16 –/dim /SSC low population (H). CD3 and CD56 were used to distinguish CD56 + NK cells, CD3 + T cells and a CD3 + CD56 + T cell population (I). The T cells were divided into CD4 + and CD8 + T cells (J). |
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