A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.
Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the "gold standard" for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.
Division of Pediatrics, Cell Therapy Section, The University of Texas MD Anderson Cancer Center, Houston, Texas, 77030, United States of America.
Srinivas S
SS
Somanchi
Department of Technology R&D, Nexcelom Bioscience LLC, 360 Merrimack St. Building 9, Lawrence, Massachusetts, 01843, United States of America.
Kelsey J
KJ
McCulley
Division of Pediatrics, Cell Therapy Section, The University of Texas MD Anderson Cancer Center, Houston, Texas, 77030, United States of America; Graduate School of Biomedical Sciences, The University of Texas Health Sciences Center, Houston, Texas, 77030, United States of America.
Anitha
A
Somanchi
Department of Technology R&D, Nexcelom Bioscience LLC, 360 Merrimack St. Building 9, Lawrence, Massachusetts, 01843, United States of America.
Leo L
LL
Chan
Division of Pediatrics, Cell Therapy Section, The University of Texas MD Anderson Cancer Center, Houston, Texas, 77030, United States of America; Graduate School of Biomedical Sciences, The University of Texas Health Sciences Center, Houston, Texas, 77030, United States of America.
Dean A
DA
Lee
D017209:Apoptosis; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D007694:Killer Cells, Natural / Q000473:pathology*; D014407:Tumor Cells, Cultured
D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM