TBMNK Cell Flow Cytometry Panel

Immunophenotyping of T Cells, B Cells, Monocytes, and NK Cells

T cells, B cells, and NK cells are the major lymphocyte subsets in peripheral blood. Monocytes are critical mediators of early host immune responses. This multicolor flow cytometry panel allows for easy identification of each cell type.



Flow Cytometry Panel for Immunophenotyping of T, B, Monocytes, and NK Cells

Marker Clone Fluorochrome Catalog #
CD3 UCHT1* Alexa Fluor® 405 FAB100V
CD4 11830 FITC FAB3791F
CD8 37006 APC FAB1509A
CD19 4G7-2E3 PE FAB4867P
CD56 2524C Alexa Fluor® 700 FAB24086N
CD16 245536 PerCP FAB2546C
CD14 134620 Alexa Fluor® 750 FAB3832S

*Designate clones independently validated by HLDA.
Alexa Fluor® is registered trademark of Molecular Probes, Inc.

This multicolor flow cytometry panel was validated on human peripheral blood mononuclear cells (PBMCs).


Flow Cytometry Gating Strategy for TBMNK Panel

Pseudocolor flow cytometry plot showing gating strategy for CD4 and CD8 T cells, CD19 B Cells, CD14+ Classical Monocytes, CD16+ Non-classical Monocytes, and CD56 NK cells.

Multicolor flow cytometry panel to identify human T Cells, B Cells, NK cells and Monocyte subsets. PBMCs were stained with anti-human CD3 Alexa Fluor® 405, CD4 FITC, CD8 APC, CD56 Alexa Fluor® 700, CD19 PE, CD14 Alexa Fluor 750, and CD16 PerCP. All antibodies are validated for flow cytometry. Classical monocytes are defined as CD14+CD16-; non-classical monocytes are CD16+CD14-; CD4 T cells are CD3+, CD4+; CD8 T cells are CD3+, CD8+; B cells are CD3-, CD19+; and NK cells are CD3-, CD56+.



Staining Protocol for TBMNK Panel

Other Supplies Required

Surface Stain

  1. Wash human PBMCs (1 x 106 cells per sample) with 2 mL of Staining Buffer (1X) (Catalog # FC001) or other BSA-containing buffer, by spinning at 300 x g for 5 minutes, using 5 mL flow cytometry tubes. Decant/aspirate supernatant.
  2. Fc-block cells with blocking IgG (1 μg IgG/106 cells) for 10 minutes at room temperature.
  3. Add previously titrated amount of each primary conjugated antibody. Vortex tubes.

    Recommended Antibody Concentrations
    Marker Fluorochrome Size Recommended Concentration
    CD3 Alexa Fluor® 405 100 tests 5 μL/106 cells
    CD4 FITC 100 tests 10 μL/106 cells
    CD8 APC 100 tests 10 μL/106 cells
    CD19 PE 100 tests 10 μL/106 cells
    CD56 Alexa Fluor® 700 100 μg 0.25-1 μg/106 cells
    CD16 PerCP 100 tests 10 μL/106 cells
    CD14 Alexa Fluor® 750 100 tests 5 μL/106 cells
  4. (Optional) To a separate tube, add 5 μL of each of the isotype control antibodies. Vortex tubes.
  5. Incubate the mixtures for 30-45 minutes at room temperature in the dark.
  6. At the end of the incubation, wash with 2 mL of Staining Buffer (1X), by spinning at 300 x g for 5 minutes. Decant/aspirate supernatant.