Evaluating Nobiletin's Enhancement of PTX Efficacy

Sulphorhodamine B (SRB) assays were used for cell density determination, based on sensitive measure of total cellular protein, which perform similarly compared with other proliferation assays such as MTT assay44 (link). Briefly, cells were seeded into flat bottomed 96-well plates at an initial density of 7.5 × 10³ per well before treatment. Cells were exposed to varying concentrations of nobiletin (9, 4.5, 1.5 and 0.5 μM) and combined them with varying concentrations of PTX (1 μM to 0.03 nM with 3.16 fold diluted, 10 μM to 0.3 nM with 3.16 fold diluted, 100 μM to 3 nM with 3.16 fold diluted respectively) to test whether this combination can enhance the growth inhibition of MDR cancer cells. After removing the medium, cells were fixed in 10% trichloroacetic acid for 1 h at 4 °C and then washed with water five times. 0.4% SRB dissolved in 1% v/v acetic acid was added and incubated 30 min for staining. The cells were quickly washed with 1% acetic acid and left to dry overnight. The protein bound SRB was solubilized by adding 200 μl 10 mM Tris buffer per well and was measured at wavelengths 490 nm using a plate reader (Spectra MAX 250; Molecular Devices, Sunnyvale, CA). The degree of resistance was estimated by comparing the IC₅₀ (concentration of 50% inhibition) for the MDR cells to that of parent sensitive cells, while, the degree of reversal of MDR was calculated by dividing the IC₅₀ for cells with the chemotherapeutic drugs in the absence of nobiletin by that obtained in the presence of nobiletin.