T lymphocyte immunophenotyping: 13-color flow cytometry panel design using the NovoCyte flow cytometer

Abstract

The human immune system is highly complex and immune status is associated with disease status, treatment efficiency, and response to external stimuli such as vaccines. For example, the immunophenotyping of leukemia has become indispensable for diagnosis of hematological malignancies. Monitoring T lymphocytes has gained diagnostic importance in the prognosis and prediction of therapies. In this study, a series of T-cell markers previously used in the HIPC (Human Immunology Phenotyping Consortium) project were assessed. Through comprehensive consideration and analysis of the expression of each marker, fluorescence intensity, spectral overlap, and NovoCyte configuration, a 13-color T-cell phenotyping panel was designed. Next, peripheral blood mononuclear cells (PBMCs) were tested on a NovoCyte. Live T cells (CD3+/Aqua−) were identified as either T helper cells (Th) or cytotoxic T cells (Tc) cells by expression of CD4 and CD8 antigens. Next, further T cell subtype was determined by CCR7 and CD45RA: naïve (CD45RA+/CCR7+), effector (TEF, CD45RA+/CCR7−), effector memory (TEM, CD45RA−/CCR7−) and central memory (TCM, CD45RA−/CCR7+). Activation of T cells was assessed by the activation marker HLA-DR. Identification of Th subtypes (Th1, Th2 and Th17) was determined by CXCR3 and CCR6. Regulatory T cells (Treg) were identified as CD4+/CD25+/CD127lo/CCR4+, and memory and naïve Treg cells were further differentiated by CD45RO. With this panel, the phenotype and activation of T cell subtypes can be determined in one run which allows quickly and in-depth analysis of human T lymphocyte subsets. NovoCyte flow cytometer features high resolution of dim signals and flexible configuration and is particularly suitable for multicolor analysis.

Pulmonary inflammation promoted by type-2 dendritic cells is a feature of human and murine schistosomiasis

“…3−6 and 12−18. For human sputum analysis, sentinel gating 69 was used to look at a numerous cell types in a 16-17 colour flow cytometry panel. Specifically, certain sentinel markers (CD3, CD16, CD45, CD11c, and CD66b) were used to specify cell populations, for instance CD3 for T cells, and were assigned a unique fluorophore and channel.…”