An In Vitro Assay for Evaluating Natural Killer Cell Cytotoxicity against Cancer Cells

Abstract

Source: Baik, C. H., et al. A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function. J. Vis. Exp. (2019)

This video demonstrates performing a lactate dehydrogenase release assay to determine the cytotoxicity of natural killer (NK) cells against the target cancer cells. The assay quantifies the release of the cytoplasmic lactate dehydrogenase enzyme from cancer cells by measuring the absorbance of the red-colored product formed.

Protocol

1. Cytotoxicity Assay: Assay Protocol

1. Use a round bottom, culture treated 96-well plate to set up the plate as suggested in Table 1. This table shows the experimental controls and 3 sets of NK experimental columns. This can be expanded to a total of 10 NK experimental columns in a 96-well plate.
2. Centrifuge the assay plate at 250 x g for 4 min to be certain that the effector and target cells are in contact. Incubate the 96-well plate for 5 hours in a humidified chamber incubator at 37 °C, 5% CO₂ to achieve ample contact between target and effector cells and target cell lysis by effector cells.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well Tissue Culture Plate	CELLTREAT	229190	Sterile, Round Bottom
Cell scraper	Fisher	8100241
Cytotox 96 Non-Radioactive Cytotoxicity Assay Kit	Promega	G1780
RPMI	Gibco	11875135	1640 Medium
YAC-1 cell	ATCC	TIB-160