Skip to main content
SpringerLink
Log in

The determination of cellular viability of hybridoma cells in microtitre plates: A colorimetric assay based on neutral red

  • Original Research Papers
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

A colorimetric assay utilising Neutral Red (C.I. 50040), a nuclear stain, was developed to determine the cellular viability of hybridoma cells in microtitre plates. A linear correlation (r=0.99) was found to exist between the uptake of Neutral Red by viable cells and the viable cell count determined by Trypan blue exclusion test. The linearity stretched over the range of cell concentrations normal in batch cultures (2–30×104/0.2 ml) with as little as ±6% intra-plate well-to-well variation and ±10.2% inter-assay variation.

Microscopical examinations of viable hybridoma cells stained with Neutral Red showed that it was located in the nucleus. The possble bifunctional activity of the Neutral Red assay as a test for cellular viability and estimating the DNA content of hybridoma cells is discussed along with its application in a drug screening programme.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD (eds.) (1983) Molecular Biology of The Cell (3rd edition), (pp. 412). Garland Publishing Inc. New York and London.

    Google Scholar 

  • Allison AC and Young MR (1969) Vital staining in fluorescence microscopy of lysosomes. In: Dingle JT and Fell HB (eds.) Volume 2 (pp. 600–626) Wiley, New York.

    Google Scholar 

  • Borenfreund E and Puerner JA (1985) Toxicity determinedin vitro by morphological alterations and Neutral Red absorption. Toxicity Letters 24: 119–124.

    Google Scholar 

  • Buckel P, Hubner-Parajsz C, Mattes R, Lenz H, Haug H and Beauchamp K. (1987) Cloning and nucleotide sequence of heavy and light chain cDNAs from a creatine-kinase-specific monoclonal antibody. Gene 51: 13–19.

    Google Scholar 

  • CloneticsTM corporation pamphlet (1990) Neutral Red bioassay.

  • Coco-Martin JM, Oberink JW, Tiny AM, van der Velden-de Groot CAM and Beuvery EC (1992) Viability measurement of hybridoma cells in suspension cultures. Cytotechnology 8: 57–64.

    Google Scholar 

  • Cook JA and Mitchell JB (1989) Viability measurements in mammalian cell systems. Analytical Biochemistry 179: 1–7.

    Google Scholar 

  • Evans HM and Schulermann W (1914) The action of vital stains belonging to the benzydene group. Science 34: 443–454.

    Google Scholar 

  • Kull FC and Cuattrecasas P (1981) Preliminary characterisation of the tumour cell cytotoxin in tumour necrosis serum. J. Immunology 126: 1279–1283.

    Google Scholar 

  • Laughton C (1984) Quantification of attached cells in microtitre plates based on Coomassie Brilliant Blue G-250 staining of total cellular protein. Analytical Biochemistry 140: 417–423.

    Google Scholar 

  • Mirabelli CK, Bartus H, Bartus JOL, Johnson R, Mong SM, Sung CP and Crooke ST (1985) Application of a tissue culture microtitre test for the detection of cytotoxic agents from natural products. J. Antibiotics 68: 758–766.

    Google Scholar 

  • Modha K (1990) An investigation into the effects of inhibitors of DNA biosynthesis on monoclonal antibody production in hybridoma cell cultures. Ph.D Thesis, University of Surrey, Guildford, Surrey.

    Google Scholar 

  • Modha K, Whiteside PJ and Spier RE (1991) Dissociation of MAB production from cell division using DNA biosynthesis inhibitors. In: Spier RE, Griffiths JB and MacDonald C (eds.) Animal Cell Technology: Developments, Processing and Products, (pp. 79–87). Butterworth-Heinemann.

  • Nilsson K, Scheirer W, Merton O-W, Ostberg L, Leihl E, Katinger HWD and Mosbach K. (1983) Entrapment of animal cells for the production of monoclonal antibodies and other biomolecules. Nature 302: 629–630.

    Google Scholar 

  • Parish CR and Mullbacher A (1983) Automated colorimetric assay for T-cell cytotoxicity. J. Immunological Methods 58: 25–237.

    Google Scholar 

  • Patterson Jr MK (1979) Measurement of growth and viability of cells in culture. Methods in Enzymology 58: 141–148.

    Google Scholar 

  • Sasaki DT, Dumas SE and Engleman EG (1987) Discrimination of viable and non-viable cells using propidium iodide in two colour immunofluorescence. Cytometry 8: 413–430.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Microbiology, Medical Sciences Building, University of Leicester, LE1 9HN, Leicester, UK

    K. Modha

  2. School of Biological Science, University of Surrey, GU2 5XH, Guildford, Surrey, UK

    J. P. Whiteside & R. E. Spier

Authors
  1. K. Modha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. P. Whiteside
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R. E. Spier
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Modha, K., Whiteside, J.P. & Spier, R.E. The determination of cellular viability of hybridoma cells in microtitre plates: A colorimetric assay based on neutral red. Cytotechnology 13, 227–232 (1993). https://doi.org/10.1007/BF00749819

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00749819

Key words

Search

Navigation