A Real‐Time Cytotoxicity Assay as an Alternative to the Standard Chromium‐51 Release Assay for Measurement of Human NK and T Cell Cytotoxic Activity

Julien Fassy1, Kyriaki Tsalkitzi1, Maria Goncalves‐Maia2, Véronique M. Braud1

1 Institut de Pharmacologie Moléculaire et Cellulaire, Université Côte d'Azur, Valbonne, 2 Institute for Research on Cancer and Aging, Université Côte d'Azur, Nice
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.42
DOI:  10.1002/cpim.28
Online Posting Date:  August, 2017
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Abstract

This unit describes the monitoring and quantification of cellular cytotoxicity using a non‐radioactive and real‐time cytotoxic assay. The extent of target‐cell lysis is monitored over time by imaging and quantifying live fluorescent target cells using a cell‐imaging multimode reader. This assay is performed in a 96 well plate in optimized culture conditions at 37°C in the presence of 5% CO2. The basic protocol describes natural killer cell‐mediated cytotoxic assay that can be adapted to include antibodies blocking inhibitory NK receptors or triggering antibody‐dependent cell‐mediated cytotoxicity (ADCC). The assay is also suitable for antigen specific T‐cell cytotoxic assays. Until now, the standard chromium 51 (51Cr)‐release assay has remained the sole sensitive assay but its major drawbacks include cost and hazard of handling radioactivity. The real‐time cytotoxic assay is therefore an effective alternative providing a robust and sensitive assay that accurately monitors lysis of target cells over time. © 2017 by John Wiley & Sons, Inc.

Keywords: cell cytotoxic assay; antibody‐dependent cell‐mediated cytotoxicity; natural killer cells; cytotoxic T cells

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Quantification of Natural Killer Cell‐Mediated Cytotoxic Activity Using a Real‐Time Digital Bio‐Imaging Assay
  • Alternate Protocol 1: Monitoring of the Regulation of NK Cell‐Mediated Cytotoxic Activity Using a Real‐Time Digital Bio‐Imaging Assay
  • Alternate Protocol 2: Measurement of ADCC Using a Real‐Time Digital Bio‐Imaging Assay
  • Alternate Protocol 3: Quantification of T Cell‐Mediated Cytotoxic Activity Using a Real‐Time Digital Bio‐Imaging Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Quantification of Natural Killer Cell‐Mediated Cytotoxic Activity Using a Real‐Time Digital Bio‐Imaging Assay

  Materials
  • K562 erythroleukemic cells (ATCC, cat. no. CCL‐243; DSMZ, cat. no. ACC‐10)
  • SUDHL4 B cell lymphoma cells (ATCC, cat. no. CRL‐2957; DSMZ, cat. no. ACC‐495)
  • OciLy19 B cell lymphoma cells (DSMZ, cat. no. ACC‐528)
  • Human PBMC: freshly isolated (see unit 7.1; Fuss et al., ) or from liquid nitrogen storage (see unit 14.3; Sierich & Eiermann, )
  • Highly purified human NK cell populations isolated by magnetic separation (see unit 7.7; Whiteside, )
  • Human NK cell lines expanded following allogenic stimulation (see unit 7.7 Whiteside, )
  • IMDM/10% FBS: complete Iscove's Modified Dulbecco's Medium ( appendix 2A) supplemented with 10% heat‐inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin
  • 500 ng/μl (500 μM) calcein‐AM cell‐permeant dye (Molecular Probes), stored at −20°C
  • 15‐ml and 50‐ml conical tubes
  • 96‐well imaging microplate with lid, black with clear flat bottom and tissue culture‐treated (Falcon)
  • Cytation 5 multi‐mode plate reader (BioTek) for cell imaging, connected to a source of CO 2, holding a GFP filter cube (Excitation 469/35/Emission 525/39, mirror 497), a 465 nm LED, and a 4× phase‐contrast objective.
  • Gen5 software (BioTek)
  • Multichannel (12‐channel) pipette with 200‐μl disposable tips
  • Additional reagents and equipment for obtaining fresh PBMC from whole blood (see units 7.1 & 14.32; Fuss et al., 7.42; Sierich & Eiermann, 7.42), frozen PBMC (see unit 14.32; Sierich & Eiermann, 7.42), highly purified human NK cells (see unit 7.7; Whiteside, 7.42), and expanded human NK cell lines (see unit 7.7; Whiteside, 7.42)
NOTE: All reagents and materials used in the preparation of the cells and during the assay must be sterile.

Alternate Protocol 1: Monitoring of the Regulation of NK Cell‐Mediated Cytotoxic Activity Using a Real‐Time Digital Bio‐Imaging Assay

  Additional Materials (also see protocol 1Basic Protocol)
  • 1 mg/ml anti‐MHC class I mAb, DX17 clone, mIgG1,K (BD Biosciences)
  • 1.1 mg/ml isotype mIgG1, MOPC 21 clone (Sigma‐Aldrich)

Alternate Protocol 2: Measurement of ADCC Using a Real‐Time Digital Bio‐Imaging Assay

  Additional Materials (also see protocol 1Basic Protocol)
  • 1 mg/ml anti‐CD20 mAb, Rituximab (Genentech) in PBS containing 0.1% BSA, prepared from a 10 mg/ml stock and stored at 4°C
  • 1.19 mg/ml human myeloma IgG1 (Sigma‐Aldrich)

Alternate Protocol 3: Quantification of T Cell‐Mediated Cytotoxic Activity Using a Real‐Time Digital Bio‐Imaging Assay

  Additional Materials (also see protocol 1Basic Protocol)
  • B‐lymphoblastoid cells immortalized by Epstein Barr virus (MHC class I type must be known)
  • 10 mM 9‐mer synthetic peptides that binds to identified MHC class I molecules, prepared in DMSO, stored at −20°C
  • Cloned human T‐cell lines (unit 7.1; Yssel & Spits, )
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Figures

Videos

Literature Cited

Literature Cited
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  Sierich, H., & Eiermann, T. (2013). Comparing individual NK cell activity in vitro. Current Protocols in Immunology, 100, 14.32.1–14.32.11. doi: 10.1002/0471142735.im1432s100.
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