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The MTT assay underestimates the growth inhibitory effects of interferons
S A Jabbar1, P R Twentyman, J V Watson
1Medical Research Council Clinical Oncology, Cambridge, UK.
Abstract:
The growth inhibitory effects of interferons, IFN-alpha and IFN-gamma on human lung cancer cell lines were studied using both a tetrazolium (MTT) colorimetric assay and direct cell counting. Significant discrepancies between the two assays were observed, the MTT assay consistently underestimating the growth inhibitory effects of the IFNs. There was no direct chemical effect of the IFNs on the tetrazolium reduction process. IFN treated cells showed increased cell size compared with control cells, although there was little or no change in cell cycle distribution. Mitochondrial activity was 30-50% greater in IFN-gamma treated cells (COR-L23) than the controls. Reduced formazan production per cell was observed in medium which had supported cell growth for several days. Differential 'medium conditioning' led to a difference in formazan production per cell between IFN and control cells and this was the major basis of the observed discrepancy. This discrepancy was not due to the differences in the glucose concentrations between these media. However, differences in pH between the media proved to be the major contributory factor of the discrepancy.
Insights
Interferons (IFNs) show growth inhibition in lung cancer cells, but the MTT assay underestimates this effect. Differences in cell-conditioned media pH explain the assay discrepancy.
Area of Science:
- Cell Biology
- Cancer Research
- Biochemistry
Background:
- Interferons (IFNs), including IFN-alpha and IFN-gamma, are cytokines with known antiproliferative effects.
- Accurate assessment of growth inhibitory effects is crucial for understanding cancer therapies.
- The tetrazolium (MTT) assay is commonly used to measure cell viability and proliferation.
Purpose of the Study:
- To investigate the growth inhibitory effects of IFN-alpha and IFN-gamma on human lung cancer cell lines.
- To compare the accuracy of the MTT assay with direct cell counting for evaluating IFN effects.
- To identify the factors contributing to discrepancies between the two assays.
Main Methods:
- Human lung cancer cell lines were treated with IFN-alpha and IFN-gamma.
- Cell growth was assessed using both the MTT colorimetric assay and direct cell counting.
- Cell size, cell cycle distribution, mitochondrial activity, and medium composition (pH, glucose) were analyzed.
Main Results:
- Significant discrepancies were observed, with the MTT assay underestimating IFN's growth inhibitory effects.
- IFN-treated cells exhibited increased cell size but similar cell cycle distribution compared to controls.
- Increased mitochondrial activity was noted in IFN-gamma treated cells; reduced formazan production per cell was linked to 'medium conditioning', primarily due to pH differences.
Conclusions:
- The MTT assay can underestimate the antiproliferative effects of interferons on lung cancer cells.
- Cell-conditioned medium pH significantly influences formazan production, causing discrepancies in MTT assay results.
- Direct cell counting provides a more reliable measure of interferon-induced growth inhibition in this context.