A Flow Cytometry Assay to Identify Immune Cell Subsets in Peripheral Blood Mononuclear Cells
Transcript
Take peripheral blood mononuclear cells or PBMCs, a mixed-cell population including lymphocytes and monocytes.
Add a fluorescent viability dye to discriminate dead cells, and a fluorescently labeled antibody cocktail to identify different cell types.
The fluorochrome A-conjugated antibodies bind to surface markers — CD3 on T cells, γδ T cell receptor or TCR on γδ T cells, and CD56 on natural killer or NK cells.
The fluorochrome B-conjugated antibodies bind to CD4 and CD8 on T cells, CD19 on B cells, and CD14 on monocytes.
Using flow cytometry, identify the live cell subsets.
While CD8+ and CD4+ T cells are CD3+, CD4+ cells lie between CD3 single-positive and CD3-CD8 double-positive cells.
γδ T cells express γδ TCRs and higher CD3 compared to CD4+ and CD8+ cells.
B cells and NK cells are CD3– but are identified by CD19 on B cells and CD56 on NK cells.
Monocytes are identified by CD14 expression.
