An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells.
The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.
Department of Immunology, Institute of Cell Biology, University of Tübingen, and German Cancer Consortium, DKFZ Partner site Tübingen, Tübingen, Germany. Electronic address: janet-peper@gmx.de.
Janet Kerstin
JK
Peper
Department of Immunology, Institute of Cell Biology, University of Tübingen, and German Cancer Consortium, DKFZ Partner site Tübingen, Tübingen, Germany. Electronic address: heiko_schuster@gmx.de.
Heiko
H
Schuster
Department of Immunology, Institute of Cell Biology, University of Tübingen, and German Cancer Consortium, DKFZ Partner site Tübingen, Tübingen, Germany; Department of General, Visceral and Transplant Surgery, University Hospital of Tübingen, Tübingen, Germany. Electronic address: markus.loeffler@uni-tuebingen.de.
Markus W
MW
Löffler
Department of Clinical and Experimental Transfusion Medicine, University Hospital of Tübingen, Tübingen, Germany. Electronic address: barbara.schmid-horch@med.uni-tuebingen.de.
Barbara
B
Schmid-Horch
Department of Immunology, Institute of Cell Biology, University of Tübingen, and German Cancer Consortium, DKFZ Partner site Tübingen, Tübingen, Germany. Electronic address: rammensee@uni-tuebingen.de.
Hans-Georg
HG
Rammensee
Department of Immunology, Institute of Cell Biology, University of Tübingen, and German Cancer Consortium, DKFZ Partner site Tübingen, Tübingen, Germany. Electronic address: stefan.stevanovic@uni.tuebingen.de.
Stefan
S
Stevanović
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000276:immunology*; D002448:Cell Adhesion / Q000276:immunology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000276:immunology; D002478:Cells, Cultured; D002860:Chromium Radioisotopes / Q000378:metabolism; D018920:Coculture Techniques; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D017097:Electric Impedance; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D009980:Influenza A virus / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000276:immunology*; D013997:Time Factors; D014763:Viral Matrix Proteins / Q000276:immunology
D002860:Chromium Radioisotopes; C524866:HLA-A*02 antigen; D015789:HLA-A2 Antigen; D010446:Peptide Fragments; D014763:Viral Matrix Proteins
Chromium release assay; Impedance; Real time; T-cell cytotoxicity assay; xCELLigence