Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

Begin with a cell pellet containing natural killer or NK cells, and fluorescently stained target cells.

Resuspend the pellet in a suitable medium and incubate.

The NK cell interacts via surface adhesion proteins, forming an immunological synapse with the target cell.

This initiates NK-cell signaling, releasing cytotoxic granules, containing perforin and granzymes into the synapse.

Perforin, a pore-forming protein, creates channels in the target cell's membrane, allowing granzymes to enter the cytoplasm.

Granzyme, a serine protease, interacts with intracellular proteins, activating the apoptotic pathway in the target cell.

Now, add propidium iodide fluorescent dye.

The dye enters dead cells through their damaged membranes and binds to the DNA by intercalating between its base pairs.

Consequently, the dead cells express double fluorochrome while the live cells express single fluorochrome.

The sample is ready for flow cytometry to assess NK cell-induced cytotoxicity by quantifying dead and live target cell numbers.

To prepare a cytotoxicity assay sample, first label 1.5-milliliter tubes for each sample and/or participant as appropriate, and pipette the desired ratio of NK cells, and DiO-labeled K562 cells into each tube.

Collect the cells by centrifugation, and carefully remove the supernatant without disturbing the cell pellet. Next, resuspend the cell mixture in 500 microliters of incomplete NK cell medium, and label the cells in each tube with 5 microliters of propidium iodide.

Collect the cells via another centrifugation, and incubate the cold culture at 37 degrees Celsius for two hours. At the end of the incubation, centrifuge the cells under the same centrifuge conditions, and resuspend the pellet in 25 microliters of fresh and complete NK cell culture medium.