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A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound

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Abstract

The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H2O2 levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the final metabolite of DCFH-DA inside a cell, and the linearity relationship between cell number and H2O2 level. The results showed that there was a perfect association between cell number and fluorescent intensity determined by the DCFH-DA method, no matter whether using resuspended or adherent cells, and further 50% concentration of inhibition (IC50) comparison between data obtained by DCFH-DA method or MTT method using a positive known anticancer compound Baicalin showed that there were no significant differences in cellular sensitivity determination to compound Baicalin though there existed a relatively higher coefficient of variation of IC50 by the DCFH-DA method than that by the MTT method. Thus our data indicate that DCFH-DA might not only be a fine reagent for determination of H2O2 levels in cells but also an ideal fluorescent dye for cellular sensitivity test of anti-cancer compounds, and may be suitable for primary high-throughput drugs screening.

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Abbreviations

MTT:

3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide

XTT:

3′-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate

MTS:

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

WST-1:

4-(3-4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzenedisulfonate

DCFH-DA:

2′,7′-dichlorfluorescein-diacetate

H2DCF:

2′,7′-dichlorodihydrofluorescin

DCF:

2′,7′-dichlorofluorescein

DMSO:

Dimethyl sulfoxide

PBS:

Phosphate-buffered saline

References

  • Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M (1983) Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 130:1910

    CAS  Google Scholar 

  • Cory AH, Owen TC, Barltrop JA, Cory JG (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 3:207–212

    CAS  Google Scholar 

  • Du G, Han G, Zhang S, Lin H, Wu X, Wang M, Ji L, Lu L, Yu L, Liang W (2010) Baicalin suppresses lung carcinoma and lung metastasis by SOD mimic and HIF-1alpha inhibition. Eur J Pharmacol 630:121–130

    Article  CAS  Google Scholar 

  • Gao J, Morgan WA, Sanchez-Medina A, Corcoran O (2011) The ethanol extract of Scutellaria baicalensis and the active compounds induce cell cycle arrest and apoptosis including upregulation of p53 and Bax in human lung cancer cells. Toxicol Appl Pharmacol 254:221–228

    Article  CAS  Google Scholar 

  • Gonzalez RJ, Tarloff JB (2001) Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity. Toxicol In Vitro 15:257–259

    Article  CAS  Google Scholar 

  • Goodwin CJ, Holt SJ, Downes S, Marshall NJ (1995) Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS. J Immunol Methods 179:95–103

    Article  CAS  Google Scholar 

  • Halliwell B, Whitemann M (2004) Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and do the results mean? Br J Pharmacol 142:231–255

    Article  CAS  Google Scholar 

  • Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241–251

    Article  CAS  Google Scholar 

  • Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K (1996) A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull 19:1518–1520

    Article  CAS  Google Scholar 

  • Marshall NJ, Goodwin CJ, Holt SJ (1995) A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. Growth Regul 5:69–84

    CAS  Google Scholar 

  • Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  CAS  Google Scholar 

  • O’Brien J, Wilson I, Orton T, Pognan F (2000) Investigation of the Alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267:5421–5426

    Article  Google Scholar 

  • Ottonello L, Frumento G, Arduino N, Dapino P, Tortolina G, Dallegri F (2001) Immune complex stimulation of neutrophil apoptosis: investigating the involvement of oxidative and nonoxidative pathways. Free Radic Biol Med 30:161–169

    Article  CAS  Google Scholar 

  • Page B, Page M, Noel C (1993) A new fluorometric assay for cytotoxicity measurements in vitro. Int J Oncol 3:473–476

    CAS  Google Scholar 

  • Rhee SG, Chang TS, Jeong W, Kang D (2010) Methods for detection and measurement of hydrogen peroxide inside and outside of cells. Mol Cells 29:539–549

    Article  CAS  Google Scholar 

  • Royall JA, Ischiropoulos H (1993) Evaluation of 2, 7-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 302:348–355

    Article  CAS  Google Scholar 

  • Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR (1988) Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48:4827–4833

    CAS  Google Scholar 

  • Takahashi H, Chen MC, Pham H, Angst E, King JC, Park J, Brovman EY, Ishiguro H, Harris DM, Reber HA, Hines OJ, Gukovskaya AS, Go VL, Eibl G (2011) Baicalein a component of Scutellaria baicalensis, induces apoptosis by Mcl-1 down-regulation in human pancreatic cancer cells. Biochim Biophys Acta 1813:1465–1474

    Article  CAS  Google Scholar 

  • Tammariello SP, Quinn MT, Estus S (2000) NADPH oxidase contributes directly to oxidative stress and apoptosis in nerve growth factor-deprived sympathetic neurons. J Neurosci 20:RC53

    Google Scholar 

  • Twentyman PR, Luscombe M (1987) A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer 56:279–285

    Article  CAS  Google Scholar 

  • Wood ZA, Poole LB, Karplus PA (2003) Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science 300:650–653

    Article  CAS  Google Scholar 

  • Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssiere JL, Petit PX, Kroemer G (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 181:1661–1672

    Article  CAS  Google Scholar 

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Acknowledgments

The authors are grateful to Dr. Dawei Li’s lab, Shanghai Jiaotong University, for providing the Varioskan Flash Multimode Microplate Reader.

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Authors and Affiliations

  1. Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240, China

    Wenwei Mao, Xinlin Chen, Tian Yang, Yu Yin, Mei Ge, Minyu Luo, Daijie Chen & Xiuping Qian

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  1. Wenwei Mao
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  2. Xinlin Chen
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  3. Tian Yang
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  4. Yu Yin
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  5. Mei Ge
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  6. Minyu Luo
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  7. Daijie Chen
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  8. Xiuping Qian
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Correspondence to Xiuping Qian.

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Mao, W., Chen, X., Yang, T. et al. A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound. Cytotechnology 64, 451–457 (2012). https://doi.org/10.1007/s10616-011-9423-0

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