TBNK Cell Flow Cytometry Panel

T cell with fluorescent green primary conjugated antibodies attached layered in front of a B cell and NK cell.

Gating Strategy | Staining Protocol | Additional Flow Cytometry Resources

Immunophenotyping of T Cells, B Cells and NK Cells

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

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

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

*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 TBNK Panel
 

Pseudocolor flow cytometry plot showing gating strategy for CD4 and CD8 T cells, CD19 B Cells, and CD56 NK cells.

Multicolor flow cytometry panel to identify human T Cells, B Cells, and NK cells. PBMCs were stained with anti-human CD3 Alexa Fluor® 405, CD4 FITC, CD8 APC, CD56 Alexa Fluor® 700, and CD19 PE. All antibodies are validated for flow cytometry. CD4 T cells are defined as 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 TBNK 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. (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.

Recommended Antibody Concentrations  

MarkerFluorochromeRecommended Concentration
CD3Alexa Fluor® 4055 μL/106 cells
CD4FITC10 μL/106 cells
CD8APC10 μL/106 cells
CD19PE10 μL/106 cells
CD56Alexa Fluor® 7000.25-1 μL/106 cells 
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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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