, Volume 17, Issue 1, pp 27–33 | Cite as

The methylene blue colorimetric microassay for determining cell line response to growth factors

  • Maria F. Dent
  • Linda Hubbold
  • Heather Radford
  • Anne P. Wilson


The validity of the methylene blue colorimetric microassay for determining the response of monolayers of human ovarian tumour cell lines to different growth factors was investigated. Linearity of the relationship between cell density and optical density was confirmed for each cell line (r=0.989–0.999,p<0.001), and when initial cell density was optimised to give exponential growth over the assay period, differences in response to medium supplements were obvious. The response of target cells to growth factors, obtained using the methylene blue assay, were compared with, and found to parallel, previously documented responses obtained non-colorimetrically. Thus Mink lung epithelial cells (MLEC) were inhibited by TGβ (Holleyet al., 1983), EGF had an inhibitory effect on A431 cells (Gill & Lazar, 1981; Barnes, 1982), and the mesothelial cell line showed a proliferative response to EGF and hydrocortisone (Connell and Rheinwald, 1983).

The methylene blue colorimetric microssay was found to be a simple, reliable, sensitive method with low variability, for determining the response of cultured cells to growth factors.

Key words

Methylene blue colorimetric microassay growth factors ovarian tumour cell lines monolayer culture 


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  1. Barnes DW (1982) Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture. Nature 93:1–4Google Scholar
  2. Connell ND & Rheinwald JG (1983) Regulation of the cytoskeleton in mesothelial cells: Reversible loss of keratin and increase in vimentin during rapid growth in culture. Cell 34:245–352.Google Scholar
  3. Denizot F & Lang R (1986) Rapid colorimetric assay for cell growth and survival. Modification to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89: 271–277.Google Scholar
  4. Fiennes AGTW, Walton J, Winterbourne D, McGlashan D & Hermon-Taylor J (1987) Quantitative correlation of neutral red dye uptake with cell number in human cancer cell cultures. Cell Biology International Reports, 11:373–378.Google Scholar
  5. Gill GN & Lazar CS (1981) Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells. Nature 293: 305–307.Google Scholar
  6. Holley RW, Armour R, Baldwin JH & Greenfield S (1983) Activity of a kidney epithelial cell growth inhibitor on lung and mammary cells. Cell Biology International Reports 7:141–147.Google Scholar
  7. Jabber SAB, Twentyman PR & Watson JV (1989) The MTT assay underestimates the growth inhibitory effects of interferons. Br. J. Cancer 60:523–528.Google Scholar
  8. Knox P, Uphill PF, Fry JR, Benford J & Balls M (1986) The FRAME multicentre project on in vitro cytotoxicology. Fd. Chem. Toxic. 24:457–463.Google Scholar
  9. Legrand C, Bour JM, Jacob C, Capiaumont J, Martial A, Marc A, Wudtke M, Kretzmer G, Demangel C, Duval D & Hache J (1992) Lactate dehydrogenase (LDH) activity of the number of dead cells in the medium of cultured eukaryotic cells as marker. J. Biotechnol. 25:231–243.Google Scholar
  10. Martin A & Clynes M (1991) Acid phosphatase: end point for in vitro toxicology tests. In Vitro Cell Dev. Biol. 27A:183–184.Google Scholar
  11. Morgan CD, Mills KC, Lefkowitz DL, & Lefkowitz SS (1991) An improved colorimetric assay for tumour necrosis factor using WEHI 164 cells cultured on novel microtiter plates. J. Immunol. Methods 58:225–237.Google Scholar
  12. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55–63.Google Scholar
  13. Parish CR & Müllbacher A (1983) Automated colorimetric assay for T cell cytotoxicity. J. Immunol. Methods 58:225–237.Google Scholar
  14. Pelletier B, Dhainaut F, Pauly A, & Zahnd J (1988) Evaluation of growth rate in adhering cell cultures using a simple colorimetric method. J. Biochemical and Biophysical Methods 16:63–74.Google Scholar
  15. Plumb JA, Milroy R & Kaye SB (1989) Effects of the pH dependence of 3-(4,5.dimethylthiazol-2-yl).2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res. 4:4435–4440.Google Scholar
  16. Racher AJ, Looby D & Griffiths JB (1990) Use of lactate dehydroge-nase release to assess changes in culture viability. Cytotechnology 3:301–307.Google Scholar
  17. 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 tumour cell lines. Cancer red. 48:4827–4833.Google Scholar
  18. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Visita D, Warren JT, Bokesch H, Kenney S & Boyd MR (1990) New colormetric cytotoxicity assay for anticancer drug screening. J. Nat. Cancer Inst. 82:1107–1112.Google Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Maria F. Dent
    • 1
  • Linda Hubbold
    • 1
  • Heather Radford
    • 1
  • Anne P. Wilson
    • 1
  1. 1.Oncology Research LaboratoryDerby City General HospitalDerbyUK

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