Assessing MTT and sulforhodamine B cell proliferation assays under multiple oxygen environments

Area of Science:

• Cell Biology
• Biochemistry
• Physiology

Background:

• Cell proliferation is crucial for biological processes.
• Assays quantifying metabolic activity or total protein are used as indirect measures of cell proliferation.
• The validity of these indirect assays under varying oxygen conditions remains unclear.

Purpose of the Study:

• To evaluate the suitability of common cell proliferation assays under different oxygen partial pressure (pO2) conditions.
• To compare cell counting with metabolic (MTT) and total protein (sulforhodamine B) assays across a range of pO2 levels.
• To determine if metabolic and protein-based assays serve as reliable surrogates for cell counting in variable oxygen environments.

Main Methods:

• Utilized permeable bottom tissue culture plates to control static oxygen concentrations (2-139 mmHg pO2).
• Measured cell proliferation via direct cell counting.
• Compared cell counts with results from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine B assays.
• Tested in Caco-2, MCF-7, MCF-10A, and PANC-1 human cell lines.

Main Results:

• MTT assay readings did not correlate with cell number in Caco-2 and PANC-1 cells across different oxygen conditions.
• Sulforhodamine B assay demonstrated consistent performance and correlation with cell number under all tested oxygen levels.
• Both MTT and sulforhodamine B assays showed correlation with cell number within a specific oxygen environment.

Conclusions:

• The MTT assay is not a reliable surrogate for cell counting when comparing cell proliferation across varying oxygen conditions.
• The sulforhodamine B assay is a more robust method for assessing cell proliferation under diverse oxygen environments.
• Caution is advised when using the MTT assay for cell growth or drug response studies involving cells cultured in different oxygen conditions.