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. 2022 Jan 22;3(1):101128.
doi: 10.1016/j.xpro.2022.101128. eCollection 2022 Mar 18.

A protocol for quantifying lymphocyte-mediated cytotoxicity using an impedance-based real-time cell analyzer

Hisashi Kanemaru  1 , Yukari Mizukami  1 , Akira Kaneko  1 , Ikko Kajihara  1 , Satoshi Fukushima  1
Affiliations

A protocol for quantifying lymphocyte-mediated cytotoxicity using an impedance-based real-time cell analyzer

Hisashi Kanemaru et al. STAR Protoc. .

Abstract

Current standard assays to analyze lymphocyte-mediated antitumor cytotoxicity employ radioisotopic or fluorescent labels. However, such assays are not suitable for real-time analysis. Here we describe a protocol that facilitates the analysis of lymphocyte-mediated toxicity using a label-free, impedance-based real-time cell analyzer. This analyzer measures cellular electrical impedance, expressed as the cell index value, noninvasively and continuously. In contrast with label-dependent assays, this protocol simultaneously generates real-time killing curves useful for quantifying lymphocyte-mediated cytotoxicity in real time. For complete details on the use and execution of this protocol, please refer to Kanemaru et al. (2021).

Keywords: Cancer; Cell Biology; Immunology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Principal of assessing lymphocyte-mediated cytotoxicity using an impedance-based real-time cell analyzer, the xCELLigence system The microtiter plates (E-Plates) of the xCELLigence system contain gold biosensors embedded in the bottom of each well. The electrical impedance of each well is reported as the cell index value, which continuously and noninvasively represents changes in cell number. If nonadherent effector cells induce the destruction of the adherent target cells, the corresponding cytolytic activity is detected. The continuous acquisition of data for each well simultaneously generates real-time killing curves under multiple conditions.
Figure 2
Figure 2
Analysis of the proliferation of target cells using the xCELLigence system (A and B) Proliferation of the indicated dilutions of target cells (B16-F1). Electrical impedance was measured and reported as the cell index value (A). Rate of change of the cell index (0–70 h) is represented by the slope (B). Similar results were obtained from three independent experiments.
Figure 3
Figure 3
Cytotoxic lymphocyte killing assay using the xCELLigence system (A and B) After adding effector cells at the indicated effector:target ratios (E:T, lymphocytes:B16-F1 cells), the system continuously measured cytotoxicity every 15 min. Wells contained 2 × 104 target cells and 100 × 104, 50 × 104, or 25 × 104 effector cells. (C and D) The cytotoxicities of lactate (Lac)-treated (20 mM) effector and control effector cells against B16-F1 target cells (E:T = 50:1) were monitored at 15-min intervals. Wells contained 2 × 104 target cells and 100 × 104 effector cells. %Cytolysis was determined using RTCA Software Light of the xCELLigence system. Three independent experiments produced similar results.
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References

    1. Cerignoli F., Abassi Y.A., Lamarche B.J., Guenther G., Santa Ana D., Guimet D., Zhang W., Zhang J., Xi B. In vitro immunotherapy potency assays using real-time cell analysis. PLoS One. 2018;13:e0193498. doi: 10.1371/journal.pone.0193498. - DOI - PMC - PubMed
    1. Delconte R.B., Kolesnik T.B., Dagley L.F., Rautela J., Shi W., Putz E.M., Stannard K., Zhang J.G., Teh C., Firth M., et al. CIS is a potent checkpoint in NK cell-mediated tumor immunity. Nat. Immunol. 2016;17:816–824. doi: 10.1038/ni.3470. - DOI - PubMed
    1. Fasbender F., Watzl C. Impedance-based analysis of Natural Killer cell stimulation. Sci. Rep. 2018;8:4938. doi: 10.1038/s41598-018-23368-5. - DOI - PMC - PubMed
    1. Hildner K., Edelson B.T., Purtha W.E., Diamond M., Matsushita H., Kohyama M., Calderon B., Schraml B.U., Unanue E.R., Diamond M.S., et al. Batf3 deficiency reveals a critical role for CD8α+ dendritic cells in cytotoxic T cell immunity. Science. 2008;322:1097–1100. doi: 10.1126/science.1164206. - DOI - PMC - PubMed
    1. Kanemaru H., Mizukami Y., Kaneko A., Tagawa H., Kimura T., Kuriyama H., Sawamura S., Kajihara I., Makino K., Miyashita A., et al. A mechanism of cooling hot tumors: lactate attenuates inflammation in dendritic cells. iScience. 2021;24:103067. doi: 10.1016/j.isci.2021.103067. - DOI - PMC - PubMed

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