Varioskan flash fluorescence plate reader

Purified LDL and HDL fractions were prepared from healthy donor plasma by ultracentrifugation (120 000 g, at 4°C, 66 h) onto a KBr layer of density 1.06 and 1.19, respectively. Fluorescent labelling was performed as previously described [19 (link)]. Briefly, 1–7 mg/ml of lipoprotein, dialyzed against phosphate-buffered saline (PBS), was incubated with Pyr-met-Chol (using ethanol as a vehicle) at 5% mol/mol of the non-esterified cholesterol amount present in the lipoprotein fraction (corresponding to 20–50 μM for HDL and 130–300 μM for LDL). Incubation was performed for 48 h at 37°C, under gentle stirring and protection from light, and labelled lipoproteins were then dialyzed using a semi-permeable membrane with a 10 kDa cutoff. Bound Pyr-met-Chol fluorescence was measured by a fluorometric assay, using a Varioskan Flash fluorescence plate reader (Thermo-Electron), with excitation and emission wavelengths at 330 nm and 400 nm respectively, against a standard curve. Protein concentration was determined by the Biorad protein assay, taking albumin as a standard.

ROS were determined as described previously [26] (link). Briefly, HT-29 cells were seeded onto 96-well plates in RPMI 1640 containing 10% FCS overnight. The next day, the medium was aspirated and replaced with fresh RPMI 1640 devoid of FCS. After 24 h, cells were treated with the ROS-sensitive DCF for 15 min, and then stimulated with EGF for indicated concentrations or for various time intervals. To assay the effect of DPI (10 µM), the drug was added to cells 20 min before the addition of EGF. The fluorescence was determined with a Varioskan Flash fluorescence plate reader (Thermo Electron Corporation, Marietta, OH, USA) with excitation at 485 nm and emission at 528 nm.

Adenosine triphosphate (ATP) concentrations, glucose consumption, and lactate secretion were measured as described in our previous report [21 (link)]. ATP concentrations were quantified with an ATP Determination Kit (Beyotime, China) using a VarioSkan flash fluorescence plate reader (Thermo Scientific, USA) according to the manufacturer’s instructions. Glucose levels in the culture medium were measured using an assay kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The lactate level in the culture medium was detected using a lactate assay kit (Biovision Inc., USA). All data were normalized by cell number.