Optimisation of Cell-seeding Density and Incubation Time for the MTS Assay in Human Oral Cell Models
DOI:
https://doi.org/10.24191/cos.v13i2.13647Keywords:
assay optimisation, cell-seeding density, MTS assay, oral cell models, oral wound healing, cellular metabolic activityAbstract
The MTS assay measures cellular metabolic activity, and its absorbance is influenced by the cell model, number of cells seeded, culture duration and incubation time after reagent addition. This study optimised cell-seeding density and MTS incubation time for human gingival fibroblasts (HGF), dental pulp stem cells (DPSC), human oral keratinocytes (HOK) and ORL-48 oral squamous cell carcinoma cells. Model-specific density series were cultured for 72 h before MTS reagent was added. Reference- and blank-corrected absorbance was measured at 490 nm, using 690 nm as the reference wavelength, at 0.5, 1, 2, 3 and 4 h after reagent addition. An investigator-defined absorbance range of 0.5-1.5 was used as a practical criterion for condition selection; it was not treated as a universal linear range. Each condition was tested in technical triplicate in three independent experiments. Absorbance increased with seeding density in all models. Descriptive linear regression yielded R² values of 0.9598 for HGF, 0.9773 for DPSC, 0.8889 for HOK and 0.8754 for ORL-48. At the 2-h measurement, 2 × 10⁴ cells/well for HGF and DPSC and 8 × 10³ cells/well for HOK and ORL-48 produced absorbance values within 0.5-1.5. Two hours was the earliest measurement at which all four models met this criterion. Subsequent mean values showed no marked increase through 4 h on descriptive visual assessment; no quantitative plateau threshold was specified. The selected conditions are specific to the tested 72-h culture and reader configuration and require separate verification at other endpoints.
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