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Inaccuracies in MTS assays: major distorting effects of medium, serum albumin, and fatty acids
Kuang Tzu Huang1, Yen Hao Chen, Ameae M Walker
1University of California, Riverside, CA 92521-0121, USA.
Abstract:
Soluble formazan assays are widely used for cell number assessment. However, in our hands, we observed frequent occasions in which the actual cell number was at odds with the assay reading. In this study, we have determined that (i) a large proportion of the reading obtained in commonly used culture media can be caused by media component amplification of formazan production in a way that cannot be corrected for by media-only controls; (ii) the albumin present in 10% serum can reduce the assay absorbance by 50% so that an actual doubling of cell number can be obscured; and (iii) this latter effect is dependent on the concentration of fatty acids. To counter these problems, we have developed a protocol that gives consistent readings that are fully representative of cell number while retaining some of the original advantages of soluble formazan assays.
Insights
Soluble formazan assays for cell counting are unreliable due to media interference and serum albumin effects. A new protocol provides accurate cell number assessment by overcoming these limitations.
Area of Science:
- Biotechnology
- Cell Biology
- Biochemistry
Background:
- Soluble formazan assays are standard for cell number determination.
- Previous studies have noted discrepancies between assay readings and actual cell counts.
Purpose of the Study:
- To investigate the causes of inaccurate readings in soluble formazan assays.
- To develop an improved protocol for reliable cell number assessment.
Main Methods:
- Investigated the influence of culture media components on formazan production.
- Assessed the impact of serum albumin and fatty acids on assay absorbance.
- Developed and validated a new protocol for soluble formazan assays.
Main Results:
- Culture media components amplify formazan production, confounding results.
- Serum albumin significantly reduces assay absorbance, obscuring cell number changes.
- Fatty acid concentration modulates the albumin-mediated reduction in absorbance.
Conclusions:
- Standard soluble formazan assays are prone to significant inaccuracies.
- A novel protocol effectively corrects for media and serum interferences.
- The improved assay provides consistent and representative cell number measurements.
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