Synergy ht multiplate reader

RNA extraction was performed with Spectrum Plant Total RNA kit (Sigma–Aldrich, St. Louis, USA), according to the manufacturer’s instructions. After the extraction, RNA samples were treated with RNase-free DNase I (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA was quantified through spectrophotometry in a Synergy HT Multiplate Reader (BioTek, Friedrichshall, Germany) with a Take3 Multi-Volume Plate (BioTek), with Gene5 software (BioTek). Sample quality and integrity were assessed through the A260/A280 ratio and through visual analysis on 2% agarose electrophoresis, respectively. Only samples with a ratio between 1.8 and 2.1 and with the two ribosomal RNA bands clearly visible were used for RNA-sequencing (RNA-seq).

Cells were seeded in 24-wells plates and cultured until 70–80% confluence. Thereafter, cells were washed with PBS and incubated with ACF or vehicle (DMSO) in supplemented serum-free RPMI 1640 medium for 24 or 48 hours. After the indicated time points, the medium was removed, cells were washed with serum- and phenol red-free RPMI 1640 medium, and cells were incubated with 100 μM DCFH₂-DA (in serum- and phenol red-free RPMI 1640 medium) for 1 hour at standard culture conditions. Next, cells were washed with serum- and phenol red-free RPMI 1640 medium, and fresh serum- and phenol red-free RPMI 1640 medium was added to the wells. Intracellular 2′,7′-dichlorofluorescein (DCF) fluorescence, which is a measure of ROS production, was read on a BioTek Synergy HT multiplate reader at λ_ex = 460 ± 40 nm and λ_em = 520 ± 520 nm. Data were obtained from n = 6 measurements and corrected for ACF fluorescence, protein content using the SRB assay, and DCF fluorescence (basal metabolic rate) of control cells.

RNA extraction was performed with Spectrum Plant Total RNA kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions, using 200 mg of powder for each sample. After the extraction, RNA samples were treated with RNase-free DNase I (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA was quantified through spectrophotometry in a Synergy HT Multiplate Reader (Biotek, Friedrichshall, Germany), using a Take3 Multi-Volume Plate (Biotek), with Gene5 software (Biotek). Sample quality was assessed through the A₂₆₀/A₂₈₀ ratio. Only samples with a ratio between 1.8 and 2.1 were chosen and submitted to RNA-sequencing (RNA-seq).