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