Determining Optimal Cell Density and Culture Medium Volume simultaneously in MTT Cell Proliferation Assay for Adherent Cancer Cell Lines

Abstract

Introduction:The MTT assay without optimization of the factors affecting cell metabolism can lead to false reports of the tested drugs. In this study, cell density and cell culture medium volume were optimized in a 96-well plate for Hepa1-6 cells as an adherent cell line. Methods: Hepa1-6 cells were seeded in the two different volumes, five cell densities, and three incubation times. After the incubation times, the optical densities were read at 570nm. The absorbances of wells in 100 and 150µl of cell culture medium volumes were compared and the optimum cell density was funded through the mean absorbance analysis of wells in the optimum volume. Results: The Independent sample t-test showed a significant difference (p-value=0.01) between the absorbances of wells with 100 and 150 µl of volumes. Also, Pearson's correlation coefficient indicated that the optimum cell culture medium was 150 µl. The optimum cell density was 5000 cells/well obtained from the liner portion of the standard curve related to the optimum volume. Discussion: The optimal cell density and volume of cell culture medium for hepa1-6 cell line were found. In addition, a simple practical protocol was suggested to find the optimal factors mentioned for the adherent cell line in MTT assay.

Synthesis, anticancer activity, and β‐lactoglobulin binding interactions of multitargeted kinase inhibitor sorafenib tosylate (SORt) using spectroscopic and molecular modelling approaches

“…After 20 min, absorbance at 630 nm was measured. [ 13,14 ] In the MTT assay, regorafenib was used as the positive control. Regorafenib, a small molecule, has a chemical structure and biochemical profile similar to that of SORt, the only difference being the addition of a fluorine atom at the centre of regorafenib, which increases the efficiency for anticancer activity.…”

Aerial Parts of Peucedanum chenur Have Anti-Cancer Properties through the Induction of Apoptosis and Inhibition of Invasion in Human Colorectal Cancer Cells

“…Viability of extract-treated HCT-116 and Vero cells was investigated using the MTT assay in a dose -and time-dependent manner in the same way. Optimum cell density was determined according to the method used by Moradi et al [ 11 ] , and 6 × 10 3 cells were seeded in each well of a 96-well plate. The cells were kept at 37 C for 24 hours, then different concentrations of methanolic extract of P. chenur (20, 60, 100, 140, 180, 220, 260, 300, 340, and 380 µg/ml) were added to each well.…”

Ensaios Colorimétricos Para Determinação Da Viabilidade Celular E Citotoxicidade in Vitro

“…No entanto, o MTT é tóxico para as células, e alguns fatores influenciam no sinal de absorbância (ver seção "Avaliação e comparação entre métodos colorimétricos"). 9,12 Este ensaio é baseado na redução do sal de tetrazólio em formazan pela oxidação de coenzimas mitocondriais (NADH e/ou FADH2) (Figura 2). No entanto, ainda há especulações sobre esse mecanismo, pois algumas pesquisas mostram que o MTT pode formar depósitos em outras organelas ou em gotículas de lipídeos.…”