Skip to main content

Cell Enumeration Assays: Application of the MTT and Sulforhodamine B Assays to Lipopolysaccharide-Stimulated Neonatal Rodent Microglia

Part of the Methods in Molecular Biology book series (MIMB,volume 1727)

Abstract

Glial cell activation, in particular microglia, plays an important role in the pathogenesis of various neurodegenerative disorders as well as in chronic and neuropathic pain. This chapter compares two established cell enumeration assays, namely, the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the protein-binding sulforhodamine B assay for microglia as a function of culture condition and activation state. The pros and cons of each are then described.

Key words

  • Microglia
  • Vitality
  • Assay
  • MTT
  • Sulforhodamine B
  • Rat
  • Cell culture
  • Activation

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Streit WJ, Mrak RE, Griffin WS (2004) Microglia and neuroinflammation: a pathological perspective. J Neuroinflammation 1:14. https://doi.org/10.1186/1742-2094-1-14

    CrossRef  PubMed  PubMed Central  Google Scholar 

  2. Miller G (2005) The dark side of glia. Science 308:778–781

    CAS  CrossRef  PubMed  Google Scholar 

  3. Slater TF, Sawyer B, Straeuli UD (1963) Studies on succinate-tetrazolium reductase systems. III Points of coupling of four different tetrazolium salts. Biochim Biophys Acta 77:383–393

    CAS  CrossRef  PubMed  Google Scholar 

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

    CAS  CrossRef  PubMed  Google Scholar 

  5. Manthorpe M, Fagnani R, Skaper SD, Varon S (1986) An automated colorimetric microassay for neuronotrophic factors. Dev Brain Res 25:191–198

    CAS  CrossRef  Google Scholar 

  6. 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 8:4827–4833

    Google Scholar 

  7. Khabar KSA, Al-Zoghaibi F, Dzimiri M, Taha M, Al-Tuwaijri A, Al-Ahdal MN (1996) MTS interferon assay: a simplified cellular dehydrogenase assay for interferon activity using a water-soluble tetrazolium salt. J Interferon Cytokine Res 16:31–33

    CAS  CrossRef  PubMed  Google Scholar 

  8. Peskin AV, Winterbourn CC (2000) A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin Chim Acta 293:157–166

    CAS  CrossRef  PubMed  Google Scholar 

  9. Vistica D, Skehan P, Scudiero DA, Monks A, Pittman A, Boyd MR (1991) Tetrazolium-based assays for cellular vitality: a critical examination of parameters which affect formazan production. Cancer Res 51:2515–2520

    CAS  PubMed  Google Scholar 

  10. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82:1107–1112

    CAS  CrossRef  PubMed  Google Scholar 

  11. Rubinstein LV, Shoemaker RH, Paull KD, Simon RM, Tosini S, Skehan P, Scudiero DA, Monks A, Boyd MR (1990) Comparison of in vitro anticancer drug screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumour cell lines. J Natl Cancer Inst 82:1113–1118

    CAS  CrossRef  PubMed  Google Scholar 

  12. Vichai V, Kirtikara K (2006) Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1:1112–1116

    CAS  CrossRef  PubMed  Google Scholar 

  13. Qin L, Wu X, Block ML, Liu Y, Breese GR, Hong JS, Knapp DJ, Crews FT (2007) Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia 55:453–462

    CrossRef  PubMed  PubMed Central  Google Scholar 

  14. Colton C, Wilcock DM (2010) Assessing activation states in microglia. CNS Neurol Disord Drug Targets 9:174–191

    CAS  CrossRef  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Stephen D. Skaper .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Facci, L., Skaper, S.D. (2018). Cell Enumeration Assays: Application of the MTT and Sulforhodamine B Assays to Lipopolysaccharide-Stimulated Neonatal Rodent Microglia. In: Skaper, S. (eds) Neurotrophic Factors. Methods in Molecular Biology, vol 1727. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7571-6_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7571-6_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7570-9

  • Online ISBN: 978-1-4939-7571-6

  • eBook Packages: Springer Protocols