Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here. It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS. The presence of SDS results in the quick de-staining of DNA and simultaneously, in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes. This increase can be attributed to the micelle formation of SDS. The method is sufficiently sensitive to reveal around 50-70 human diploid cells. It is compatible with immunocytochemical detections, the detection of DNA replication and cell cycle analysis by image cytometry. The procedure was successfully tested for the analysis of cytotoxicity. The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells. The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature. Only around 90 to 120 minutes is required for the procedure's completion.

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University in Olomouc Olomouc 779 00 Czech Republic

Zobrazit více v PubMed

Quent VMC, Loessner D, Friis T, Reichert JC, Hutmacher DW. Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research. J. Cell. Mol. Med. 2010;14:1003–1013. doi: 10.1111/j.1582-4934.2010.01013.x. PubMed DOI PMC

Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. PubMed DOI

Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 2005;11:127–152. doi: 10.1016/S1387-2656(05)11004-7. PubMed DOI

van Tonder A, Joubert AM, Cromarty AD. Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Res. Notes. 2015;8:47. doi: 10.1186/s13104-015-1000-8. PubMed DOI PMC

Khabar KSA, et al. MTS interferon assay: A simplified cellular dehydrogenase assay for interferon activity using a water-soluble tetrazolium salt. J. Interf. Cytok. Res. 1996;16:31–33. doi: 10.1089/jir.1996.16.31. PubMed DOI

Peskin AV, Winterbourn CC. A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1) Clin. Chim. Acta. 2000;293:157–166. doi: 10.1016/S0009-8981(99)00246-6. PubMed DOI

Scudiero DA, et al. Evaluation of a Soluble Tetrazolium Formazan Assay for Cell-Growth and Drug Sensitivity in Culture Using Human and Other Tumor-Cell Lines. Cancer Res. 1988;48:4827–4833. PubMed

O’Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 2000;267:5421–5426. doi: 10.1046/j.1432-1327.2000.01606.x. PubMed DOI

Zachari MA, et al. Evaluation of the alamarblue assay for adherent cell irradiation experiments. Dose Response. 2014;12:246–258. doi: 10.2203/dose-response.13-024.Koukourakis. PubMed DOI PMC

Ng KW, Leong DTW, Hutmacher DW. The challenge to measure cell proliferation in two and three dimensions. Tissue Eng. 2005;11:182–191. doi: 10.1089/ten.2005.11.182. PubMed DOI

Jones LJ, Gray M, Yue ST, Haugland RP, Singer VL. Sensitive determination of cell number using the CyQUANT (R) cell proliferation assay. J. Immunol. Methods. 2001;254:85–98. doi: 10.1016/S0022-1759(01)00404-5. PubMed DOI

Myers MA. Direct measurement of cell numbers in microtitre plate cultures using the fluorescent dye SYBR green I. J. Immunol. Methods. 1998;212:99–103. doi: 10.1016/S0022-1759(98)00011-8. PubMed DOI

Papadimitriou E, Lelkes PI. Measurement of cell numbers in microtiter culture plates using the fluorescent dye Hoechst 33258. J. Immunol. Methods. 1993;162:41–45. doi: 10.1016/0022-1759(93)90405-V. PubMed DOI

McCaffrey TA, Agarwal LA, Weksler BB. A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro. In Vitro Cell Dev. Biol. 1988;24:247–252. doi: 10.1007/BF02623555. PubMed DOI

Kapuscinski J. DAPI: a DNA-specific fluorescent probe. Biotech. Histochem. 1995;70:220–233. doi: 10.3109/10520299509108199. PubMed DOI

Sinha R, Hossain M, Kumar GS. Interaction of Small Molecules with Double-Stranded RNA: Spectroscopic, Viscometric, and Calorimetric Study of Hoechst and Proflavine Binding to PolyCG Structures. DNA Cell Biol. 2009;28:209–219. doi: 10.1089/dna.2008.0838. PubMed DOI

Wilson WD, Ratmeyer L, Zhao M, Strekowski L, Boykin D. The Search for Structure-Specific Nucleic-Acid Interactive Drugs - Effects of Compound Structure on Rna Versus DNA Interaction Strength. Biochemistry-Us. 1993;32:4098–4104. doi: 10.1021/bi00066a035. PubMed DOI

Tanious FA, Veal JM, Buczak H, Ratmeyer LS, Wilson WD. Dapi (4′,6-Diamidino-2-Phenylindole) Binds Differently to DNA and Rna - Minor-Groove Binding at at Sites and Intercalation at Au Sites. Biochemistry-Us. 1992;31:3103–3112. doi: 10.1021/bi00127a010. PubMed DOI

Kapuscinski J, Skoczylas B. Fluorescent complexes of DNA with DAPI 4′,6-diamidine-2-phenyl indole.2HCl or DCI 4′,6-dicarboxyamide-2-phenyl indole. Nucleic Acids Res. 1978;5:3775–3799. doi: 10.1093/nar/5.10.3775. PubMed DOI PMC

Gorner H. Direct and sensitized photoprocesses of bis-benzimidazole dyes and the effects of surfactants and DNA. Photochem. and Photobiol. 2001;73:339–348. doi: 10.1562/0031-8655(2001)0730339DASPOB2.0.CO2. PubMed DOI

Cifuentes A, Bernal JL, DiezMasa JC. Determination of critical micelle concentration values using capillary electrophoresis instrumentation. Anal. Chem. 1997;69:4271–4274. doi: 10.1021/ac970696n. DOI

Landry JJM, et al. The Genomic and Transcriptomic Landscape of a HeLa Cell Line. G3-Genes Genom. Genet. 2013;3:1213–1224. PubMed PMC

Macville M, et al. Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res. 1999;59:141–150. PubMed

Adan A, Kiraz Y, Baran Y. Cell Proliferation and Cytotoxicity Assays. Curr. Pharm. Biotechnol. 2016;17:1213–1221. doi: 10.2174/1389201017666160808160513. PubMed DOI

Erba E, Ubezio P, Broggini M, Ponti M, D’Incalci M. DNA damage, cytotoxic effect and cell-cycle perturbation of Hoechst 33342 on L1210 cells in vitro. Cytometry. 1988;9:1–6. doi: 10.1002/cyto.990090102. PubMed DOI

Brenan M, Parish CR. Intracellular fluorescent labelling of cells for analysis of lymphocyte migration. J. Immunol. Methods. 1984;74:31–38. doi: 10.1016/0022-1759(84)90364-8. PubMed DOI

Ellwart JW, Dormer P. Vitality measurement using spectrum shift in Hoechst 33342 stained cells. Cytometry. 1990;11:239–243. doi: 10.1002/cyto.990110204. PubMed DOI

Myc A, Traganos F, Lara J, Melamed MR, Darzynkiewicz Z. DNA stainability in aneuploid breast tumors: comparison of four DNA fluorochromes differing in binding properties. Cytometry. 1992;13:389–394. doi: 10.1002/cyto.990130409. PubMed DOI

Blaheta RA, et al. Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity. J. Immunol. Methods. 1998;211:159–169. doi: 10.1016/S0022-1759(97)00202-0. PubMed DOI

Sandhu LC, Warters RL, Dethlefsen LA. Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA. Cytometry. 1985;6:191–194. doi: 10.1002/cyto.990060304. PubMed DOI

Cosa G, Focsaneanu KS, McLean JR, McNamee JP, Scaiano JC. Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution. Photochem. Photobiol. 2001;73:585–599. doi: 10.1562/0031-8655(2001)073<0585:PPOFDD>2.0.CO;2. PubMed DOI

Godinho, C. P. et al. Pdr18 is involved in yeast response to acetic acid stress counteracting the decrease of plasma membrane ergosterol content and order. Sci. Rep. 8 (2018). PubMed PMC

Barcellona ML, Cardiel G, Gratton E. Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides. Biochem. Biophys. Res. Commun. 1990;170:270–280. doi: 10.1016/0006-291X(90)91270-3. PubMed DOI

Singer VL, Jones LJ, Yue ST, Haugland RP. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal. Biochem. 1997;249:228–238. doi: 10.1006/abio.1997.2177. PubMed DOI

Freshney, R. I. Cytotoxicity in Culture of Animal Cells. A manual of basic techniques. (ed. Freshney, R. I.) 365–369 (John Wiley and Sons, Inc., 2005).

Ligasová A, et al. Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine. Open Biol. 2016;6:150172. doi: 10.1098/rsob.150172. PubMed DOI PMC

Ligasová A, Strunin D, Friedecký D, Adam T, Koberna K. A fatal combination: a thymidylate synthase inhibitor with DNA damaging activity. PLoS One. 2015;10:e0117459. doi: 10.1371/journal.pone.0117459. PubMed DOI PMC

Carpenter AE, et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7:R100. doi: 10.1186/gb-2006-7-10-r100. PubMed DOI PMC

Kamentsky L, et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics. 2011;27:1179–1180. doi: 10.1093/bioinformatics/btr095. PubMed DOI PMC

Ligasová A, Strunin D, Liboska R, Rosenberg I, Koberna K. Atomic Scissors: A New Method of Tracking the 5-Bromo-2 ‘-Deoxyuridine-Labeled DNA In Situ. Plos One. 2012;7:e52584. doi: 10.1371/journal.pone.0052584. PubMed DOI PMC

A Rapid Approach for Identifying Cell Lines Lacking Functional Cytidine Deaminase

The kinetics of uracil-N-glycosylase distribution inside replication foci

A new technique for the analysis of metabolic pathways of cytidine analogues and cytidine deaminase activities in cells

DNA Dyes-Highly Sensitive Reporters of Cell Quantification: Comparison with Other Cell Quantification Methods