Viability Assays

Cells were seeded in 96-well plates and left to attach for 24 h. Dead and unattached cells were washed with PBS while the remaining viable cells were further exposed for 24 h/48 h to the HI extract. Following the exposure, the cells were washed with PBS and viability was assessed by two complementary assays, Alamar Blue assay, and Neutral Red assay.

Alamar Blue (AB) assay was used to measure the metabolic ability of exposed cells to convert resazurin, a non-fluorescent compound, to resorufin, a fluorescent product. The cells were exposed to a resazurin solution of 200 μM for 3 h and the fluorescence was measured at λexcitation = 530/25, λemission = 590/35, using Synergy 2 multi-mode microplate reader.

Neutral Red (NR) assay was used to measure the ability of the exposed cells to incorporate the supravital dye neutral red. The cellular uptake of this dye reveals the capacity of the cell to maintain pH gradients through the production of ATP. Briefly, 150 μL 40 μg/mL neutral red dye was added to each well. The cells were incubated with the dye at 37 ◦C for 2 h. Afterwards, the cells were washed twice with PBS and accumulated dye was extracted by adding a solution containing 50% ethanol, 49% water, and 1% glacial acetic acid to each well. Cell viability was determined by measuring the fluorescence at λexcitation = 530/25, λemission = 620/40, using Synergy 2 multi-mode microplate reader.

The experiments were performed with three biological replicates, each one including six technical replicates. The results were expressed as relative values compared to the negative control (100%) (cells exposed to culture medium containing 0.2% DMSO).

IC50 values were calculated, when possible, from the dose-response curves obtained for each condition using a four-parameter logistic curve, in order to allow a comparison between the conditions tested.
