title: A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells.
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.
authors.affiliation: Department of Clinical Pharmacology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Makiko
authors.initials: M
authors.lastname: Yamashita
authors.affiliation: Department of Experimental Therapeutics, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.|Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Shigehisa
authors.initials: S
authors.lastname: Kitano
authors.affiliation: Department of Clinical Pharmacology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Aikawa
authors.affiliation: Division of Biostatistics, Center for Research Administration and Support, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Kuchiba
authors.affiliation: Department of Clinical Pharmacology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.|Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Hayashi
authors.affiliation: Department of Experimental Therapeutics, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.|Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.|Division of Breast and Medical Oncology, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Tamura
authors.affiliation: Department of Clinical Pharmacology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.|Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Akinobu
authors.initials: A
authors.lastname: Hamada
mesh_terms: D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D000954:Antigens, Surface / Q000378:metabolism; D001943:Breast Neoplasms / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D015925:Cryopreservation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000378:metabolism*; D010641:Phenotype
chemical_list: D000954:Antigens, Surface
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