Method Article

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

July 8th, 2025

In This Article

Abstract

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Source: Torchia, M. L. G., et al. Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry. J. Vis. Exp. (2019)

This video demonstrates a flow cytometry-based technique using two fluorochromes to identify CD4+ and CD8+ T cells, γδ T cells, B cells, natural killer (NK) cells, and monocytes in human peripheral blood mononuclear cells (PBMCs). Upon incubating the cells with fluorochrome-conjugated antibodies against the surface markers, the cell subsets are identified using an optimal flow cytometric gating strategy.

Protocol

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All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

NOTE: This protocol has been tested on freshly or frozen isolated peripheral blood cells and whole blood.

1. Cell Staining

NOTE: Choosing pairs of fluorochromes with virtually no spectral overlap is important to reduce the spread of data due to the high spillover of a fluorochrome in the other fluorochrome detector. To achieve an optimal identification of all the cell subsets, fluorochromes with a high quantum yield should be....

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Results

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Flow cytometry analysis showing CD8+ cell staining at varying dilutions, table of stain indices, and optimal antibody concentration graph.
Figure 1: Representative antibody titration. (A) The dot plot shows CD8 expression on fresh PBMC stained with the indicated concentration of the antibody. (B) The table represents t.......

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
CD3BD Biosciences562426Antibody for staining
RRID: AB_11152082
CD56BD Biosciences562751Antibody for staining
RRID: AB_2732054
TCRgdBD Biosciences331217Antibody for staining
RRID: AB_2562316
CD4BD Biosciences555347Antibody for staining
RRID: AB_395752
CD8BD Biosciences555367Antibody for staining
RRID: AB_395770
CD14BD Biosciences555398Antibody for staining
RRID: AB_395799
CD19BD Biosciences555413Antibody for staining
RRID: AB_395813
CD3BD Biosciences555335Antibody for staining
RRID: AB_398591
CD56BD Biosciences555518Antibody for staining
RRID: AB_398601
TCRgdBD Biosciences331211Antibody for staining
RRID: AB_1089215
CCR7Biolegend353217Antibody for staining
RRID: AB_10913812
CD45RABD Biosciences560674Antibody for staining
RRID: AB_1727497
CCR4BD Biosciences561034Antibody for staining
RRID: AB_10563066
CCR6BD Biosciences353419Antibody for staining
RRID: AB_11124539
CXCR3BD Biosciences561730Antibody for staining
RRID: AB_10894207
CD57BD Biosciences562488Antibody for staining
RRID: AB_2737625
HLA-DRBD Biosciences563083Antibody for staining
RRID: AB_2737994
CD16BD Biosciences563784Antibody for staining
RRID: AB_2744293
Dead cellsLife technologiesL-23105Live/dead discrimination
96-well V-bottom plateThermo fisher249570plate for staining
FACSAria IIu Cell SorterBD BiosciencesFlow cytometer
FCS Express 6De Novo SoftwareFACS analysis
Graphpad PrismGraphPad softwareData analysis

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Tags

Two fluorochrome AssayCell Surface MarkersFluorescent AntibodiesGating StrategyCD4 T CellsCD8 T CellsNatural Killer Cells

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