A Novel Method for Evaluating Antibody-dependent Cell-mediated Cytotoxicity by Flowcytometry Using Cryopreserved Human Peripheral Blood Mononuclear Cells

Overview

Journal Sci Rep

Specialty Science

Date 2016 Jan 28

PMID 26813960

Citations 29

Authors

Makiko Yamashita

Shigehisa Kitano

Hiroaki Aikawa

Aya Kuchiba

Mitsuhiro Hayashi

Noboru Yamamoto

Kenji Tamura

Akinobu Hamada

Affiliations

Soon will be listed here.

Abstract

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The (51)Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies.

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