MBTP 35: Immunophenotyping of tumor-infiltrating lymphocytes from human ovarian carcinoma using flow cytometry 

Miltenyi Biotec-tested panel 35 (MBTP 35)

This application protocol describes the characterization of human monocytes, neutrophils, eosinophils, and T, B, and NK lymphocyte populations as well as CD4+, CD8+, and CD56+CD3+ T cell subsets in human ovarian carcinoma using flow cytometry. Prior to staining the tumor was dissociated employing the gentleMACS™ Octo Dissociator with Heaters and Tumor Dissociation Kit, human.

Protocol

Gating strategy showing the analysis of the immune cell composition in a dissociated human ovarian carcinoma sample. A gate around viable cells (A) as well as a gate around single cells in forward scatter area (FSC-A) versus forward scatter height (FSC-H) (B) was set to eliminate doublets. To identify the major circulating blood cell types CD45 was used to target all leukocytes (C). These cells were further separated from debris via FSC and side scatter (SSC) (D). Monocytes were discriminated based on their CD14 expression (E). Among the non-monocyte population, B cells were defined as CD19+ (F). The remaining cells were separated into CD16+ SSChigh neutrophils, CD16 SSChigh eosinophils as well as a CD16–/dim SSClow population (G). CD3 and CD56 were used to distinguish CD56+ NK cells, CD3+ T cells and a CD3+CD56+ T cell population (H). CD3+ T cells were divided into CD4+ helper T cells and CD8+ cytotoxic T cells (I).

Materials

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