Calcein Acetoxymethyl Staining to Evaluate Natural Killer Cell-Mediated Cytotoxicity toward Cancer Cells

1. Calcein AM Staining-based Microscopic Method for Measuring NK Cell-mediated Cytotoxicity

1. Culture SK-HEP-1 cells to 70−80% confluency. Generate a single-cell suspension by first washing cells with 5 mL of 1x PBS followed by incubation with 1 mL of 0.25% trypsin-EDTA.
2. Centrifuge SK-HEP-1 cells in a 1 mL sterile centrifuge tube at 160 x g for 3 min. Resuspend the pellet in 3 mL of serum-free DMEM.
3. Add 1.5 µL of calcein AM solution (10 mM) to SK-HEP-1 cells and incubate for 30 min at room temperature. Centrifuge calcein AM-labeled SK-HEP-1 cells at 160 x g for 3 min in a 15 mL sterile centrifuge tube.
4. Wash cells twice with 5 mL of 1x PBS to remove excess calcein AM dye.
5. In parallel, centrifuge NK92MI cells at 160 x g for 3 min in a 15 mL sterile centrifuge tube. Wash the cell pellet once with 5 mL of 1x PBS and resuspend in 5 mL of NK92MI cell medium.
6. Count calcein AM-labeled SK-HEP-1 cells and NK92MI cells using a hemocytometer or an automated cell counter.
7. Resuspend SK-HEP-1 cells in the culture medium at 1 x 10⁵ cells/mL and NK92MI cells at 1 x 10⁶ cells/mL in the NK cell medium.
8. Plate calcein AM-labeled SK-HEP-1 cells (target cells) (1 x 10⁴/100 µL/well) with NK92MI cells (effector cells) (1 x 10⁵/100 µL/well) (1:10 target: effector ratio) per well in triplicate wells in a 96 well plate.
9. Incubate the 96-well plates at 37 °C in an atmosphere of 95% air and 5% CO₂ for 4 h. After incubation, capture fluorescence images of the calcein AM-labeled cells using a fluorescence microscope at 10x magnification. Capture at least 10 different fields of each replicate for each treatment condition.
10. Randomly select 10 images for each replicate and count calcein AM-positive labeled target cells incubated with or without NK92MI cells. Calculate % cytotoxicity using the formula below.
    
    NOTE: As controls, use target cells without NK92MI cells and completely lysed target cells as complete lysis control. For complete lysis, incubate the cells in 0.5% Triton X-100 for 1 h (20 µL of 5% Triton X-100 in 200 µL of culture media).