Intracellular NK Cell Activation Flow Cytometry Panel

Gating Strategy | Staining Protocol | Additional Flow Cytometry Resources

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 NK Cell Activation Markers

*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 Intracellular NK Cell Activation Panel

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 PerCP, Granzyme K Alexa Fluor^® 594, Granzyme B Alexa Fluor^® 488, Perforin Alexa Fluor^® 750, CD56 Alexa Fluor^® 647, and CD3 Alexa Fluor^® 405. 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

• PBS
• Flow Cytometry Staining Buffer (Catalog # FC001)
• Flow Cytometry Fixation Butter (Catalog # FC004)
• Flow Cytometry Permeabilization Buffer/Wash Buffer I (Catalog # FC005)
• Fc-block (blocking IgG)
• (Optional) Isotype Control Antibodies
• 5 mL Flow cytometry tubes

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 surface marker. Vortex tubes.
   
   NK Cell Activation Antibody Concentration Chart  

   Marker	Fluorochrome	Recommended Concentration
   CD3	Alexa Fluor® 405	5 μL/106 cells
   NCAM1/CD56	Alexa Fluor® 647	0.25-1 μg/106 cells
   Fc Gamma R III (CD16)	PerCP	10 μL/106 cells

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 previously titrated amount of conjugated antibody to intracellular activation markers. Vortex tubes.
   
   NK Cell Activation Antibody Concentration Chart

   Marker	Fluorochrome	Recommended Concentration
   Granzyme B	Alexa Fluor® 488	5 μL/106 cells
   Granzyme K	Alexa Fluor® 594	0.1-1 μg/106 cells
   Perforin-1	Alexa Fluor® 750	0.25-1 μg/106 cells

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.

Additional Flow Cytometry Products and Resources