2 7 dichlorofluorescein

PNT2, LNCaP and PC-3 cells seeded in 24-well black plates were incubated with HDL, sHDL or apoA-I at 0.5 mg/ml up to 16 h in RPMI 1640 (supplemented with 1% bovine serum albumin for LNCaP). Cells were then washed with PBS and loaded with 3 μM 2′,7′-dichlorofluorescein (Life Technologies, USA) for 30 minutes. After RPMI 1640 replenishment, cells were stimulated or not with 0.5 mM H₂O₂ for 1 h and ROS production was measured by fluorescence on a Synergy H1 multi-mode reader equipped with the Gen5 software (BioTek, USA). To evaluate the role of AR signalling in ROS generation and HDL antioxidant effect, in separate experiments LNCaP were maintained in RPMI 1640 without phenol red and incubated overnight with the AR antagonist bicalutamide (100 nM, Sigma-Aldrich). To evaluate the role of cell cholesterol content on HDL antioxidant effect, cells were pre-treated overnight with LDL (50 μg/ml) or for 1 h with 2.5 mM beta-methylcyclodextrin (βMCD, Sigma Aldrich, Germany).
For each sample, fluorescence was normalized by the protein concentration of the total cell lysate, measured by the micro-bicinchoninic acid assay (Thermo Scientific, USA).
A standard HDL preparation from pooled plasma was tested in each experiment for inter-assay data correction.

QH and OE33 cells were seeded at 15,000 cells per well in a 96-well cell culture plate, reverse transfected as described above and allowed to incubate for 24 h. Cell supernatant was removed and the cells were washed with 100 μL PBS/Mg²⁺ buffer (130 mM NaCl, 5 mM KCl, 1 mM Na²PO₄, 1 mM CaCl₂, 1 mM MgCl₂ and 25 mM HEPES, pH 7.4) and subsequently incubated for 30 min at 37 °C in PBS/Mg²⁺ containing 5 µM 2′,7′-dichlorofluorescein (Invitrogen), 0.3 µM mitotracker (Invitrogen, California, USA) or 5 µM rhodamine 123 (Sigma, St. Louis, MO, USA) to assess reactive oxygen species production, mitochondrial mass and mitochondrial membrane potential respectively. 2′,7′-dichlorofluorescein and mitotracker were solubilized in DMSO, whereas rhodamine 123 was solubilized in ethanol. Cells were washed with 200 μL PBS/Mg²⁺ buffer, the buffer discarded, 100 μL fresh buffer added and fluorescence read with excitation and emission wavelengths of 485 nm and 538 nm respectively (Thermo Scientific, Fluoroskan Ascent FL, Waltham, MA, USA). All measurements were normalized to cell number using the crystal violet assay (Sigma) (9).