Fluroskan ascent fl

1*10⁶ cultured cells were harvested in 200 μl PBS and cell lysate was achieved by sonication (in ice-water bath) before the homogenate was centrifugated at 12,000 g for 10 min. Finally, in 96-well plates, 10 μl of the supernatant was mixed well with 100 μl of luciferase reagent, which catalyzed the light production from ATP and luciferin ATPliteTM Luminescence Assay kit (PerkinElmer) according the manufacturer's instructions. Luminescence was measured by luminometer (Fluroskan Ascent FL, Thermo). Standard curve was also generated and the amount of ATP in the experiments samples was calculated. Total ATP levels were expressed as nmol/10⁶ cell.

Cellular lipid peroxidation was measured using C₁₁-BODIPY_581/591 (D3861 Invitrogen, Eugene, Oregon, United States) according to the manufacturer’s instructions. Briefly RGC5, HepG2 and HEK293 cells were seeded at a density of 4*10⁴, 5*10⁴ and 5*10⁴ cells/well respectively in black 96 well culture plates. Due to limited attachment, poly-l-lysine (0.01%) coated black 96 well culture plates were used for HEK293 cells. After 24 h the cells were washed once with 100 μL of PBS and were loaded with 100 μM C₁₁-BODIPY_581/591 in HBSS for 30 min under normal culture conditions. Cells were washed once with 100 μL PBS before treated with different concentrations of CQ (0, 0.5, 1, 5, 10 μM) in HBSS for different time intervals (0, 30, 60, 90 and 120 min). After the treatment period, cells were washed once with 100 μL PBS and fluorescence was quantified immediately in 40 μL PBS (Ex/Em 460/535 nm: green fluorescence and 485/600 nm: red fluorescence) using a multimode plate reader (Fluroskan Ascent FL, Thermo Scientific, VIC, Australia). Changes in fluorescence intensity ratio at 535 nm versus 600 nm, indicative of lipid peroxidation, was expressed as a percentage of untreated control values. Lipid peroxidation was measured in at least three replicate wells/sample/experiment. Three independent experiments were performed with one representative experiment shown.

1 × 10⁶ cultured cells were harvested in 200 μl PBS and cell lysate was achieved by sonication (in ice-water bath) before the homogenate was centrifuged at 12,000 g for 10 min. The intracellular ATP production was assessed by using a bioluminescence assay (firefly luciferase) ATP Determination Kit (A22066, Molecular Probes™) and the manufacturers' instructions were strictly followed. Luminescence was measured by a luminometer (Fluroskan Ascent FL, Thermo Scientific). Data were normalized based on the protein concentration measured by the Brandford assay. Total ATP levels were expressed as nmol/mg protein.

Glycated BSA was carried out according to our previous study [15 (link)]. The mixture of bovine serum albumin (BSA, 10 mg/mL), isolated compound (0.1 mM in dimethyl sulfoxide, DMSO), and methylglyoxal (MG, 0.25 mM) in 0.1 M phosphate buffer solution (PBS, pH 7.4) was incubated at 37 °C for 24 h. The glycated BSA was monitored by a spectrofluorometer (Fluroskan Ascent FL, Thermo Scientific, Barrington, IL, USA) at the excitation wavelength of 355 nm, and an emission wavelength of 460 nm. Aminoguanidine hydrochloride (AG) was utilized as a positive control. The percentage inhibition of MG-derived AGE formation was calculated by utilizing the formula below: $\mathrm{Inhibition}\mathrm{of}\mathrm{AGEs}\mathrm{formation}(\%)=\frac{\left(\mathrm{FC}-\mathrm{FCB}\right)-\left(\mathrm{FS}-\mathrm{FSB}\right)}{\mathrm{FC}-\mathrm{FCB}}\times 100$
where FC and FCB are the fluorescent intensities of the control with and without MG, respectively. FS and FSB are the fluorescent intensities of the sample with and without MG, respectively.

Portions (10 μl) of the lipid mixture of E. coli total lipid extract were dried and rehydrated with 50 mM CF, 100 mM sucrose, and 5 mM HEPES/KOH (pH 7.4). Multilamellar liposomal suspensions were extruded with a 0.1-μm polycarbonate membrane using an Avanti Mini Extruder and purified by a PD-10 column (GE Healthcare, Buckinghamshire, United Kingdom) as previously described. The peptides were added to LUVs, and CF leakage was measured using Fluroskan Ascent FL (Thermo Labsystems, UK) in external buffer (150 mM KCl and 10 mM HEPES/Tris; pH 7). CF leakage was calculated using the following formula: CF leakage (%) = 100 × (F – F₀)/(F_max – F₀), where F is the measured fluorescence intensity, F₀ is the basal LUV fluorescence intensity, and F_max is the fluorescence intensity of LUVs treated with 0.2% Triton X-100.