Novostar plate reader

PRP was loaded with Fura-2 AM (2 µM) for 1 h at 30 °C and then washed platelets were prepared. Fura-2AM-loaded platelets were incubated with PXR ligands or vehicle prior to their activation. The ratio of emission values (excitation at 340/380 nm) was recorded using a NOVOstar plate reader (BMG Labtech) and converted to calcium concentration. Detailed method is available in the supplementary information.

The production of ROS in isolated mitochondrial samples was tested using the MitoSOX Stain (ThermoFisher, United Kingdom). Isolated mitochondria were added to a black-walled 96 well plate, spiked with MitoSOX Red (final concentration = 5 µM) and incubated for 15 min in the dark at 37°C 5% CO₂. Changes in MitoSOX fluorescence (excitation 510/emission 580 nm) were measured using the BMG Labtech NovoStar plate reader. A baseline was measured for 16 s, at which point antimycin (final concentration 244 µM) or MAB was injected into appropriate wells. Fluorescence was then measured for a further 234 s (total time = 250 s).

Primary MEFs were loaded by endocytosis with fluorescein-dextran (pH sensitive) and Texas Red-dextran (pH insensitive) at 0.2 mg/ml in growth medium in 96-well plates at 37°C for 16 h. The cells were washed three times with dextran-free medium and immediately processed. Fluorescence measurements were collected from labeled endo-lysosomes using a Novostar plate reader (BMG Labtech) using excitation/emission of 485/520 nm for fluorescein and 570/620 nm for Texas Red. Autofluorescence (cells not loaded with dextrans) was subtracted from the fluorescein and Texas Red fluorescence. Fluorescein fluorescence (G) was divided by the Texas Red fluorescence (R), and the pH was determined against a standard curve obtained in the following way: dextran-loaded MEFs were equilibrated for at least 10 min in a high-K⁺ extracellular buffer (5 mM NaCl, 145 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose) and adjusted to a series of defined pH values in buffers (10 mM acetate for pH 4 to 5, 10 mM MES [morpholineethanesulfonic acid] for pH 5.5 to 6.5, and 10 mM HEPES for pH 7) containing 2 μM nigericin and 2 μM valinomycin. Fluorescence measurements were acquired as described above. Fluorescein- and Texas Red-dextrans (molecular weight, 10,000) were purchased from Invitrogen.

Serum ACE activity was measured as described by Beneteau et al [12 (link)]. ACE activity was determined with an artificial substrate (FAPGG, (N-[3-(2-furyl) acryloyl]-L-phenylalanyl-glycylglycine; Sigma-Aldrich) in a reaction mixture containing 25 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 0.5 mM FAPGG, 300 mM NaCl, and the desired dilution of serum at pH 8.2. Measurements were performed in 96-well plates (Greiner-Bio One) at 37 °C. Changes in optical density (340 nm) were measured at 5-min intervals for at least 90 min with a plate reader (NovoStar plate reader; BMG Labtech). Optical density values were plotted as a function of reaction time and fitted by linear regression (GraphPad Prism 5.0). The fit and the data were accepted when r² was >0.90. ACE activity was calculated with the equation: $\mathit{activity}=\left(S/k\right)D$ where S is the rate of observed decrease in optical density (1/min), k is the change in optical density upon the complete cleavage of 1 μmol of FAPGG, and D is the dilution of the serum. ACE activity is given in units where 1 U is equivalent to the cleavage of 1 μmol of FAPGG in 1 min. The reference range for serum ACE level was between 8 and 52 U/L for adults, and between 13 and 100 U/L for children less than 18 years of age.