Cell density and solvent are critical parameters affecting formazan evaluation in MTT assay

Author(s)

Kellen Cristina da Silva Gasque ^[1] ; Luana Polioni Al-Ahj ^[1] ; Rodrigo Cardoso Oliveira ^[1] ; Ana Carolina Magalhães ^[4]

Affiliation

^[1] Univ Sao Paulo, Fac Odontol Bauru, Dept Ciencias Biol, Sao Paulo - Brazil

^[2] Universidade de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciências Biológicas - Brasil

• Document type: Journal article
• Source: Brazilian Archives of Biology and Technology; v. 57, n. 3, p. 381-385, 2014-02-25.

Abstract

The aim of this study was to establish the more accurate protocol for fibroblast cell viability using MTT assay. NIH/3T3 fibroblasts were seeded at the following cell densities: 3.125x10³; 1.156x10(4); 3.125x10(4); 1.156x10(5) and 3.125x10(5) cells/cm². Following 24h of seeding, MTT was added to the wells. After 4h of the MTT addition, different solvents were added to solubilize the formazan crystals: 1) HCl/SDS group- 20% SDS and 0.01 M HCl; 2) EtOH/ HAc group-50% ethanol and 1% acetic acid; 3) DMSO group- 99.5% dimethyl sulfoxide; and 4) PropOH group- 99.5% isopropanol. The absorbance values were measured using a spectrophotometer at 570 nm. The data were analyzed by 2-way ANOVA (p<0.05) and showed that the absorbance average varied according to the number of cells and solvents: HCl/SDS (0 to 0.13), EtOH/HAc (0 to 0.22), DMSO (0.76 to 1.31) and PropOH (0.66 to 1.04). The DMSO and PropOH groups presented the most appropriate protocols for NIH/3T3 fibroblasts cell viability, especially at the density of 1.156x10(4) cells/cm². (AU)

• FAPESP's process: 10/14984-4 - EFFECT OF FLUORIDE SALTS ON THE VIABILITY OF FIBROBLASTS AND PRE-OSTEOBLASTS
• Grantee: Ana Carolina Magalhães
• Support Opportunities: Regular Research Grants
• FAPESP's process: 09/17360-4 - Effect of Titanium Tetrafluoride on viability and morphology of NIH3T3 fibroblasts
• Grantee: Luana Polioni Al-Ahj
• Support Opportunities: Scholarships in Brazil - Scientific Initiation