This video demonstrates an assay to measure the cytotoxicity of natural killer cells against cancer cells using the Calcein AM stain. The reduced number of fluorescent cancer cells following co-culture with natural killer cells indicates natural killer cell-mediated cytotoxicity toward cancer cells.
Protocol
1. Calcein AM Staining-based Microscopic Method for Measuring NK Cell-mediated Cytotoxicity
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.
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.
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.
Wash cells twice with 5 mL of 1x PBS to remove excess calcein AM dye.
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.
Count calcein AM-labeled SK-HEP-1 cells and NK92MI cells using a hemocytometer or an automated cell counter.
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.
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.
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.
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).