TBMNK Cell Flow Cytometry Panel

Identification of T cells, B cells, NK cells, and monocytes with primary conjugated antibodies.

Gating Strategy | Staining Protocol | Additional Flow Cytometry Resources

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

MarkerCloneFluorochromeCatalog #
CD3UCHT1*Alexa Fluor® 405FAB100V
CD411830FITCFAB3791F
CD837006APCFAB1509A
CD194G7-2E3PEFAB4867P
CD562524CAlexa Fluor® 700FAB24086N
CD16245536PerCPFAB2546C
CD14134620Alexa Fluor® 750FAB3832S

*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

Multicolor flow cytometry panel to identify human T Cells, B Cells, NK cells and Monocyte subsets.

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+.

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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.

  4. Recommended Antibody Concentrations

    MarkerFluorochromeSizeRecommended Concentration
    CD3Alexa Fluor® 405100 tests5 μL/106 cells
    CD4FITC100 tests10 μL/106 cells
    CD8APC100 tests10 μL/106 cells
    CD19PE100 tests10 μL/106 cells
    CD56Alexa Fluor® 700100 μg0.25-1 μg/106 cells
    CD16PerCP100 tests10 μL/106 cells
    CD14Alexa Fluor® 750100 tests5 μL/106 cells
  5. (Optional) To a separate tube, add 5 μL of each of the isotype control antibodies. Vortex tubes.
  6. Incubate the mixtures for 30-45 minutes at room temperature in the dark.
  7. 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.
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Additional Flow Cytometry Products and Resources

Products:

Isotype Controls
MagCellect™ Cell Selection Kits
Quality Control and Standardization Beads from Novus Biologicals

Resources:

Flow Cytometry Handbook
Intracellular Staining with Alcohol Permeabilization Protocol
Intracellular Staining with Detergent Permeabilization Protocol
On-Demand Webinar: Demystifying Multi-parameter Flow Cytometry
On-Demand Webinar: Turning Flow Cytometry Upside Down and Inside Out

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