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An improved MTT assay

D Sladowski¹, S J Steer, R H Clothier

¹FRAME Alternatives Laboratory, Department of Human Morphology, University of Nottingham Medical School, Queen's Medical Centre, UK.

Journal of Immunological Methods

|January 4, 1993

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This study introduces a modified MTT assay using MultiScreen filtration plates for improved cell viability and proliferation measurements. The enhanced method increases accuracy and reproducibility by allowing direct optical density measurement on the plate.

Area of Science:

Biotechnology
Cell Biology
Assay Development

Background:

The MTT assay is a common method for assessing cell viability and proliferation.
Traditional MTT assays can face challenges with reproducibility and accuracy, particularly in removing culture medium.
Standard methods may lead to loss of cells or formazan crystals during solubilization.

Purpose of the Study:

To develop a modified MTT assay with enhanced reproducibility and accuracy.
To adapt the MTT assay for use with MultiScreen filtration plates.
To evaluate the performance of the modified assay using specific cell lines.

Main Methods:

The modified MTT assay was performed on MultiScreen filtration plates.
Culture medium was removed prior to formazan solubilization, preventing cell or crystal loss.

Related Experiment Videos

A 1:1 mixture of DMSO and ethanol was used as a solvent with matching optical refraction index to the filters.

Optical densities were measured directly on the MultiScreen plate.

Two cell lines, CTLL-2 (murine T cell) and Jurkat E.6.1 (human lymphoma), were used for comparison.

The modified assay was used to assess the impact of varying Interleukin-2 (IL-2) concentrations on CTLL-2 cell proliferation.

Main Results:

The modified MTT assay on MultiScreen plates demonstrated improved accuracy and reproducibility compared to standard flat-bottomed plates.
The method successfully prevented the loss of cells and formazan crystals during the assay.
Direct optical density measurements on the MultiScreen plate were feasible due to the solvent's optical properties.
The assay effectively evaluated the dose-dependent effects of IL-2 on CTLL-2 cell proliferation.

Conclusions:

The modified MTT assay using MultiScreen filtration plates offers a more reliable and accurate method for cell viability and proliferation studies.
This technique simplifies the assay procedure by enabling direct measurement on the filtration plate.
The enhanced MTT assay is suitable for evaluating cellular responses to growth factors like IL-2.