Intracellular NK Cell Activation Flow Cytometry Panel

Immunophenotyping of Intracellular NK Cell Activation

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 investigate the expression of intracellular proteins expressed during NK cell activation.

 

 

Flow Cytometry Antibodies for Intracellular Activation Markers in NK Cells

Marker Clone Fluorochrome Catalog #
CD3 UCHT1* Alexa Fluor® 405 FAB100V
NCAM1/CD56 2524C Alexa Fluor® 647 FAB24086R
Granzyme B 351927 Alexa Fluor® 488 IC296G
Granzyme K - Alexa Fluor® 594  - 
Perforin-1 1031751 Alexa Fluor® 750 IC103853S
Fc Gamma R III (CD16) 245536 PerCP FAB2546C

*Designate clones independently validated by HLDA.

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

 

 

Flow Cytometry Gating Strategy for Intracellular NK Cell Activation Panel

Pseudocolor flow cytometry plot showing gating strategy for intracellular NK Cell markers Perforin, Granzyme K, Granzyme B and surface markers CD56 and CD16.

Intracellular NK Cell 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 and 13 for expression of human CD16, Granzyme K, Granzyme B, Perforin, CD56, and CD3. Cells were gated on singlets (FSC-A by FSC-H) and live cells and quadrants were set using isotype controls (not shown).

 

 

 

Staining Protocol for Intracellular NK Cell Activation 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 5 μL (or previously titrated amount) of each surface marker (CD3 Alexa Fluor® 405, CD56 Alexa Fluor® 647, and CD16 PerCP). 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.

Intracellular Stain (with detergent permeabilization)

  1. Add 0.5 mL of cold Flow Cytometry Fixation Buffer (Catalog # FC004) and vortex. Incubate at room temperature for 10 minutes. Vortex cells intermittently to maintain a single-cell suspension.
  2. Centrifuge cells 350-500 x g for 5 minutes. Decant the Fixation Buffer.
  3. Wash cells PBS (or HBSS) by adding 2 mL of PBS (or HBSS), centrifuge at 350-500 x g for 5 minutes, and decant buffer from pelleted cells. Repeat (2 total washes).
  4. Resuspend the cell pellet in 0.1-0.2 mL of Flow Cytometry Permeabilization Buffer/Wash Buffer I (Catalog # FC005)

    Note: Saponin-mediated cell permeabilization is a reversible process, it is important to keep the cells in the presence of Permeabilization Buffer I during intracellular staining. 
    Note: Depending on the specific antibody and cell sample being used, the fixation and permeabilization steps can be performed simultaneously using Flow Cytometry Fixation/Permeabilization Buffer I (Catalog # FC007).

     
  5. Add 5-10 µL/106 cells (or a previously titrated amount) of conjugated antibody to intracellular activation markers (Granzyme B Alexa Fluor® 488, Perforin Alexa Fluor® 750, and Granzyme K Alexa Fluor® 594). Vortex tubes.
  6. Incubate cells for 30 minutes at room temperature in the dark.
  7. Wash cells 2 times with Flow Cytometry Permeabilization Buffer/Wash Buffer I (Catalog # FC005) as described in step 3.
  8. Resuspend the cells in 0.2-0.5 mL PBS (or HBSS) buffer and acquire on a Flow Cytometer.