Spectrafluor plus

Cells were seeded at 1 × 10⁵/mL in 24-well plates in DMEM supplemented as described above. After seeding (24 h), cells were treated for 24 h with ascorbic acid 3 and 30 μM, 20-hydroxyecdysone 1.5 and 15 μM, ferulic acid 1 and 10 μM, 2-hydroxycinnamic acid 2 and 20 μM, p-coumaric acid 0.1 and 1 μM, β-carotene 1.5 and 15 μM, lutein 2 and 20 μM, with the phytochemicals mixture (at their lowest concentration) and with spinach extracts at 1–5–10%. After 24 h of treatment, cells were washed with PBS and pre-incubated for 30 min (37 °C) in the dark with DCFH-DA 10 μM diluted in PBS (pH 7.4). Cells were washed with PBS to remove extracellular DCFH-DA, resuspended in DMEM, and treated 30 min at 37 °C with menadione 100 μM, a known oxidant agent [20 (link)]. The medium was removed and a lysis solution (Tris-HCL 50 mM, 0.5% TritonX pH 7.4; cell dissociation solution, Sigma Aldrich, St. Louis, MO, USA) was added for 10 min. Cell lysates were scraped from the dishes and the extracts were centrifuged. The supernatant was collected, and the fluorescence was read with a fluorescence spectrophotometer (Spectra Fluor Plus, Tecan Group Ltd., Männedorf, Switzerland) looking at the fluorescence peak between 510 and 550 nm. Each experiment was performed in triplicate.

MSCs were seeded on 96-well white plates to obtain 90% of confluence in each time point and the ATP Lite Luminescence assay kit was performed according to manufacturer’s instructions (Perkin Elmer). Luminescence was measured using the SpectraFluor Plus (Tecan Group Ltd) microplate reader.

Intracellular ROS production was monitored by the permeable fluorescence dye, 2′, and 7′-dichlorodihydrofluorescein diacetate (H2DCFDA). Interaction between H2DCFDA and ROS form the fluorescent product 2,7-dichlorofluorescein (DCF). The intracellular fluorescence intensity of DCF is proportional to the amount of ROS generated by the cells. 1.5 × 10⁵ BS-24-1 cells were incubated with 20–25 μM of H2DCFDA for 30 min at 37 °C, under 5% CO₂ followed by the addition of different concentrations of 122.4 and of the chemotherapeutic drugs. The fluorescence intensity of intracellular DCF (excitation 485 nm, emission 535 nm) was measured using a spectrofluorometer (TECAN, SPECTRAFLUOR PLUS, Tecan Group Ltd. Männedorf, Switzerland). 50 μM of the ROS-inducer, Tertiary butyl hydroperoxide (TBHP), was used as a positive control.