Resource Summary Report  New Search Previous Search Results

URL: https://dx.doi.org/DOI:10.17504/protocols.io.6j4hcqw

Authors: Elinor Gottschalk, Bulent Arman Aksoy, Pinar Aksoy, Jeff Hammerbacher

Group: Hammer Lab

Summary: A plate-based assay to estimate the cytotoxic activity of pmel-1 T cells against target cancer cells. The CytoTox-ONE Homogeneous Membrane Integrity Assay from Promega (Madison, WI) is a lactate dehydrogenase (LDH) release-based assay. Dying cells have damaged cell membranes causing cytosolic components, such as LDH, to leak into the culture media. The CytoTox-ONE assay uses LDH in the culture media to convert resazurin to fluorescent resofurin. The resulting fluorescent signal is proportional to the number of non-viable cells in a sample. One advantage to using this assay for T cell-mediated cytotoxicity is that the cancer cells do not need to be removed from the wells to measure cell death, which is especially convenient with adherent cancer cell types that would require trypsinization. However, we have found that the assay produces noisy data and a set of control wells with T cells alone is required to subtract background, overall increasing the number of replicates that are necessary. We had consistent results when culturing the pmel-1 T cells with hgp100 peptide-pulsed MC38 colon cancer cells. But we had inconsistent results using pmel-1 T cells and their natural target, B16-F10 melanoma cells. This setup could be adapted for T cells other than pmel-1 and their target cells. The ratio of T cells to cancer cells and the duration of co-culture would have to be optimized.

Affiliations: Medical University of South Carolina, Medical University of South Carolina, Medical University of South Carolina, Medical University of South Carolina

Version: 1

Publication Date: 2019

Expand All Collapse