NK Cell Phenotype Flow Cytometry Panel

Immunophenotyping of NK Cells

Natural Killer (NK) cells are innate lymphocytes that are critical for host defense against viral infections and cancer. Use this validated flow cytometry panel to for immunophenotyping of NK cell markers.

 

 

Flow Cytometry Panel for Immunophenotyping of NK Cells

Marker Clone Fluorochrome Catalog #
CD3 UCHT1* Alexa Fluor® 405 FAB100V
NCAM1/CD56 2524C Alexa Fluor® 647 FAB24086R
NKp30/NCR3 210845* Alexa Fluor® 488 FAB1849G
NKG2D/CD314 149810* Alexa Fluor® 700 FAB139N
NKp46/NCR1 195314* PE FAB1850P
Fc Gamma R III (CD16) 245536 PerCP FAB2546C

*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).

 

 

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.