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Erschienen in: Molecular Imaging and Biology 3/2018

01.06.2018 | Research Article

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay

verfasst von: Marina A. Kalinina, Dmitry A. Skvortsov, Maria P. Rubtsova, Ekaterina S. Komarova, Olga A. Dontsova

Erschienen in: Molecular Imaging and Biology | Ausgabe 3/2018

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Abstract

Purpose

High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required.

Procedures

The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system.

Results

For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format.

Conclusion

The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.
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Literatur
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Metadaten
Titel
Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay
verfasst von
Marina A. Kalinina
Dmitry A. Skvortsov
Maria P. Rubtsova
Ekaterina S. Komarova
Olga A. Dontsova
Publikationsdatum
01.06.2018
Verlag
Springer International Publishing
Erschienen in
Molecular Imaging and Biology / Ausgabe 3/2018
Print ISSN: 1536-1632
Elektronische ISSN: 1860-2002
DOI
https://doi.org/10.1007/s11307-017-1152-0

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