A Flow Cytometry Assay to Identify Immune Cell Subsets in Peripheral Blood Mononuclear Cells

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.