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Quantification of Natural Killer Cell-Mediated Cytotixicity using Celigo Imaging Cytometry
Poster Title: Quantification of Natural Killer Cell-Mediated Cytotixicity using Celigo Imaging Cytometry
Submitted on 27 Apr 2015
Author(s): Leo L. Chan, Srinivas S. Somanchi, Kelsey Rosbach, Dean A. Lee
Affiliations: Nexcelom Bioscience LLC, The University of Texas MD Anderson Cancer Center
This poster was presented at AACR 2015
Poster Views: 1,637
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Poster Information
Abstract: Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolyticT lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells (effector), they release the entrapped labels into the media upon lysis. The amount of labels in the media is measured to determine the level of cytotoxicity the effectors have induced. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel cytotoxicity assay using the Celigo imaging cytometry method. Utilizing imaging cytometry, direct cell counting of live fluorescent target cellscan be performed, which is a direct method for assessment of cytotoxicity. Human NK cells from one healthy donor were used as effectors, and K562 (suspension) and IMR32 (adherent) were used as the target cells. Both targetcells were first stained with Calcein AM, and seeded at 10,000 cells/well in a standard 96-well microplate. The donor NK cellswere then added to each well at Effector-to-Target (E:T) ratios 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125:1. The 96 well plate was then scanned and analyzed using Celigo imaging cytometer at t = 1, 2, 3, and 4 h to measure the % lysis of target cells. The results showed increasing % lysis as incubation time and E:T ratio increased. The proposed Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity, which can be an attractive method for both academic and clinical research.Summary: Time-course tracking of % lysis eliminates multiple controls & the effect of non-uniform cell seeding in the final cytotoxicity calculation. The # of cells used is less than the cells needed for Release assays & Flow Cytometry. Flow cytometry & Release assays require a seeding density of 100K target cells increasing the number of effector cells to the millions. The visual observation of ADCC or CDC on the images can be convincing to conclude the functionality of antibodies or complements.Report abuse »
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