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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References
Brimacombe KR, Hall MD, Auld DS, Inglese J, Austin CP, Gottesman MM, Fung KL (2009) A dual-fluorescence high-throughput cell line system for probing multidrug resistance. Assay Drug Dev Technol 7(3):233–249. https://doi.org/10.1089/adt.2008.165
Kang J, Hsu CH, Wu Q, Liu S, Coster AD, Posner BA, Altschuler SJ, Wu LF (2016) Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines. Nat Biotechnol 34(1):70–77. https://doi.org/10.1038/nbt.3419
Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G (2011) Cell death assays for drug discovery. Nat Rev Drug Discov 10(3):221–237. https://doi.org/10.1038/nrd3373
Sumantran VN (2011) Cellular chemosensitivity assays: an overview. Methods Mol Biol 731:219–236. https://doi.org/10.1007/978-1-61779-080-5_19
Fritzsche M, Mandenius CF (2010) Fluorescent cell-based sensing approaches for toxicity testing. Anal Bioanal Chem 398(1):181–191. https://doi.org/10.1007/s00216-010-3651-6
Enterina JR, Wu L, Campbell RE (2015) Emerging fluorescent protein technologies. Curr Opin Chem Biol 27:10–17. https://doi.org/10.1016/j.cbpa.2015.05.001
Wolff M, Wiedenmann J, Nienhaus GU, Valler M, Heilker R (2006) Novel fluorescent proteins for high-content screening. Drug Discov Today 11(23-24):1054–1060. https://doi.org/10.1016/j.drudis.2006.09.005
Guzman C, Bagga M, Kaur A et al (2014) ColonyArea: an ImageJ plugin to automatically quantify colony formation in clonogenic assays. PLoS One 9(3):e92444. https://doi.org/10.1371/journal.pone.0092444
Ferrari M, Fornasiero MC, Isetta AM (1990) MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods 131(2):165–172. https://doi.org/10.1016/0022-1759(90)90187-Z
Osterman IA, Komarova ES, Shiryaev DI, Korniltsev IA, Khven IM, Lukyanov DA, Tashlitsky VN, Serebryakova MV, Efremenkova OV, Ivanenkov YA, Bogdanov AA, Sergiev PV, Dontsova OA (2016) Sorting out antibiotics’ mechanisms of action: a double fluorescent protein reporter for high-throughput screening of ribosome and DNA biosynthesis inhibitors. Antimicrob Agents Chemother 60(12):7481–7489. https://doi.org/10.1128/AAC.02117-16
Veselov MS, Sergiev PV, Osterman IA et al (2015) [Common features of antibacterial compounds: an analysis of 104 compounds library]. Biomed Khim 61(6):785–790. https://doi.org/10.18097/pbmc20156106785
Hellweg CE, Baumstark-Khan C, Horneck G (2001) Enhanced green fluorescent protein as reporter protein for biomonitoring of cytotoxic effects in mammalian cells. Anal Chim Acta 427(2):191–199. https://doi.org/10.1016/S0003-2670(00)01021-7
Ettinger A, Wittmann T (2014) Fluorescence live cell imaging. Methods Cell Biol 123:77–94. https://doi.org/10.1016/B978-0-12-420138-5.00005-7
Kain SR (1999) Green fluorescent protein (GFP): applications in cell-based assays for drug discovery. Drug Discov Today 4(7):304–312. https://doi.org/10.1016/S1359-6446(99)01330-6
Baumstark-Khan C, Palm M, Wehner J, Okabe M, Ikawa M, Horneck G (1999) Green fluorescent protein (GFP) as a marker for cell viability after UV irradiation. J Fluoresc 9(1):37–43. https://doi.org/10.1023/A:1020583623407
Hellweg CE, Baumstark-Khan C, Rettberg P, Horneck G (2001) Suitability of enhanced green fluorescent protein as a reporter component for bioassays. Anal Chim Acta 426(2):175–184. https://doi.org/10.1016/S0003-2670(00)00824-2
Steff AM, Fortin M, Arguin C, Hugo P (2001) Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: an assay amenable to high-throughput screening technologies. Cytometry 45(4):237–243. https://doi.org/10.1002/1097-0320(20011201)45:4<237::AID-CYTO10024>3.0.CO;2-J
Page B, Page M, Noel C (1993) A new fluorometric assay for cytotoxicity measurements in-vitro. Int J Oncol 3(3):473–476
Wylie PG, Onley DJ, Hammerstein AF, Bowen WP (2015) Advances in laser scanning imaging cytometry for high-content screening. Assay Drug Dev Technol 13(2):66–78. https://doi.org/10.1089/adt.2014.607
Michael S, Auld D, Klumpp C, Jadhav A, Zheng W, Thorne N, Austin CP, Inglese J, Simeonov A (2008) A robotic platform for quantitative high-throughput screening. Assay Drug Dev Technol 6(5):637–657. https://doi.org/10.1089/adt.2008.150
Marques-Gallego P, den Dulk H, Backendorf C et al (2010) Accurate non-invasive image-based cytotoxicity assays for cultured cells. BMC Biotechnol 10(1):43. https://doi.org/10.1186/1472-6750-10-43
Acknowledgements
We thank Dr. Y.N. Schulze and Dr. L. Shaw for critical reading of the manuscript. We thank Dr. D.A. Khochenkov and Ms. M.N. Evseeva for consultations about high-throughput microscopes and to Dr. E.S. Shilov for consultations about high-throughput flow cytometer.
Funding
This work was supported by the Russian Science Foundation [RSF 14-24.00061] and Lomonosov Moscow State University Development Program [PNR 5.13].
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Kalinina, M.A., Skvortsov, D.A., Rubtsova, M.P. et al. Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay. Mol Imaging Biol 20, 368–377 (2018). https://doi.org/10.1007/s11307-017-1152-0
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DOI: https://doi.org/10.1007/s11307-017-1152-0