Multiplexing Phenotype and Function for More Biologically Relevant Insights - 14

APPLICATION NOTE

A High-Throughput, Radioactivity-Free Assay For Cell-Mediated Cytotoxicity
Introduction
Immunotherapy promises to be a powerful approach for treating a variety of
diseases-most notably cancer.1-5 However, development of novel immunotherapeutics has been limited by the lack of high-throughput methods to screen for
effective molecular entities or, in the case of adoptive cell therapy, genetically engineered cells. Most current assays that measure cell-mediated cytotoxicity (CMC),
such as the chromium release assay, are difficult to perform on large numbers
of samples, can only report on a single biological readout like cell membrane
integrity, and cannot differentiate between effector and target cells. Methods
based on flow cytometry, such as the CFSE assay,6 can assess CMC at the level of
an individual cell and enable discrimination between effector cells and target cells.
Traditional flow cytometry is slow, however, making it unsuitable for use as a high
throughput screening (HTS) assay.
Here we demonstrate a fast, efficient, radioactivity-free CMC assay with low sample
input requirements, enabling miniaturization to 384-well plates. Using the the
Intellicyt® iQue Screener and differential labeling of effector and target cells, this
method can capture multiple facets of biology-apoptosis markers, signal transduction markers, cell permeability, proliferative capability, and more-in a single
well, providing a rich and highly quantitative data set specific to each cell type
present. With this approach, researchers and drug discovery teams can quickly
screen through compounds and conditions, building a detailed understanding of
the molecular events occurring in each well and speeding insight into development of immunotherapeutic approaches.
Materials and Methods
Cells and Reagents: Jurkat cells, clone E6.1 (TIB-152 from ATCC), were used as
target cells and NK-92 cells (CRL-2407 from ATCC) were used as effector cells.
Jurkat cells were maintained in log growth phase at 37°C with 5% CO2 in 1640
RPMI medium supplemented with 10% fetal bovine serum (Seradigm). NK-92 cells
were cultured at 37°C with 5% CO2 in MyeloCult medium (Stem Cell Technologies)
supplemented with 20 ng/ml rhIL2 (Sigma) and 1 μM hydrocortisone (Sigma).
14

| January, 2019

Development of novel immunotherapeutics
has been limited by the lack of highthroughput methods to screen for effective
molecular entities...



Multiplexing Phenotype and Function for More Biologically Relevant Insights

Table of Contents for the Digital Edition of Multiplexing Phenotype and Function for More Biologically Relevant Insights

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Multiplexing Phenotype and Function for More Biologically Relevant Insights - 1
Multiplexing Phenotype and Function for More Biologically Relevant Insights - Contents
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