NK Cell Phenotype Flow Cytometry Panel

Immunophenotyping of NK Cells with primary conjugated antibodies.

Gating Strategy | Staining Protocol | Additional Flow Cytometry Resources

Immunophenotyping of 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 NK Cells

MarkerCloneFluorochromeCatalog #
CD3UCHT1*Alexa Fluor® 405FAB100V
NCAM1/CD562524CAlexa Fluor® 647FAB24086R
NKp30/NCR3210845*Alexa Fluor® 488FAB1849G
NKG2D/CD314149810*Alexa Fluor® 700FAB139N
NKp46/NCR1195314*PEFAB1850P
Fc Gamma R III (CD16)245536PerCPFAB2546C

*Designate clones independently validated by HLDA.

The NK Cell Phenotype Panel is also available as a multicolor flow cytometry kit (Catalog # FMC033) complete with isotype controls and staining buffer.

Flow Cytometry Gating Strategy for NK Cell Phenotype Panel

Pseudocolor flow cytometry plot showing gating strategy for NK Cell markers CD56, CD16, NKp30, NKp46, and NKG2D.

NK cell phenotypic analysis over a 14-day time course. NK cells were expanded from PBMCs using plate-bound Anti-Human NKp46 Monoclonal Antibody (Catalog # MAB1850) in ExCellerate™ Human NK Cell Expansion Media, Xeno-Free (Catalog # CCM032) plus rhIL-2 (27 ng/mL; Catalog # 202-IL), rhIL-12 (10 ng/mL; Catalog # 219-IL), rhIL-18 (10 ng/mL; Catalog # 9124-IL), rhIL-21 (10 ng/mL; Catalog # 8879-IL) for 13 days. NK cells were assessed on Days 0, 5, 9, and 13 for expression of human NKp30, CD16, NKG2D, NKp46, CD56 and CD3. Cells were gated on singlets (FSC-A by FSC-H) and live cells (using the Live-or-Dye 405/545 dead cell exclusion dye) and quadrants were set using isotype controls (not shown).

Back to Top

Staining Protocol for NK Cell Phenotype Panel

Other Supplies Required

  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 5 μL of each of the fluorochrome conjugated antibodies. 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.
  7. Resuspend the cells in 0.2-0.5 mL Staining Buffer (1X) and acquire on a Flow Cytometer.
Back to Top

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
Cell Markers Guide for Human Immune Cell Characterization by Flow Cytometry Poster
Interactive Cell Marker Tool
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

Back to Top