Fluoroscan ascent

H₂O₂ generation was estimated fluorimetrically at 37 °C using an Amplex Red detection system with a Thermo Scientific Fluoroscan Ascent plate reader, as described before [22 (link)] with 5 mM glutamate + 5 mM malate as substrates in the medium for kidney mitochondrial respiration [27 (link)]. The effect (changes in H₂O₂ production (in %)) was calculated according to the formula: $$Effect (\%)=\left(\frac{H_2 O_2 production with flavonoids\times 100}{H_2 O_2 production without flavonoids}\right)-100,$$

To quantify ROS we used DCFH-DA. Through intracellular esterase and ROS the non-fluorescence stain DCFH is converted to the fluorescent stain DCF. Cells were loaded with 10 mM DCFH-DA in a humidified incubator of 37°C, 95% air and 5% CO₂ for 15 min. After replacing the medium, 50 µM H₂O₂ was added for 1 h in order to generate ROS. The fluorescence was measured by using the microplate reader fluoroscan ascent (Thermo scientific) with excitation/emission wavelengths of 485/538 nm. The fluorescence was expressed as a percentage of the control cells treated only with 50 µM H₂O₂. The ROS-level was normalized by measuring the viability of the cells in the same well. An unpaired student's t-test calculated with Statistica was used to compare the data obtained. A p-value <0.05 is described as significant and a p-value <0.01 as highly significant.

In part of the muscle biopsy obtained at rest maximal enzyme activity of citrate synthase (CS), 3-Hydroxyacyl CoA dehydrogenase (HAD), phosphofructokinase (PFK), and lactate dehydrogenase (LDH) was quantified in muscle homogenates after freeze drying and removal of fat and connective tissue using fluorometric methods (Fluoroscan Ascent, Thermo Scientific, Waltham, MA) (Lowry and Passonneau 1972 ).

MPO activity was measured to determine the possible mechanism of action of NDS27. The SIEFED method allows the capture of MPO from biological fluids by microplate-coated specific antibodies. The fluid is eliminated by washings and the in situ activity of the enzyme is determined by a combination of a fluorogenic substrate (Amplex red) and a nitrite-based amplifier system. The primary antibody (rabbit anti-MPO IgG) was coated onto black microplate wells. Equine MPO standards (ranging from 0.25 to 6.4 mU/mL) and non-diluted samples containing MPO (100 μL) were added to the microplate and incubated for 2 hours at 37°C. After 3 washings, the peroxidase activity of MPO was detected by adding 100 μL of 40 μM Amplex red (10-acetyl-3, 7-dihydroxyphenoxazine), freshly prepared in phosphate buffer (50 mM) at pH 7.5 containing 10 μM H₂O₂ and 10 mM nitrite. Fluorescence was measured with a fluorescence plate reader (Fluoroscan Ascent, Fischer Scientific) at the excitation and emission wavelengths of 544 and 590 nm. Each sample was run in duplicate. The fluorescence value is directly proportional to the quantity of active MPO in the sample.

CS activity was used as an indication of mitochondrial content. CS has been validated as strong predictor of mitochondrial content in skeletal muscle(Larsen et al., 2012 (link)) and has also been used in endothelial cell cultures and arteries (Broniarek et al., 2016 (link); Park et al., 2018 (link)). Cells were lysed in a 0.3 mol/L phosphate BSA buffer adjusted to pH 7.7 and analyzed for the maximal enzyme activity of citrate synthase (CS) using a fluorometric method (Fluoroscan Ascent, Thermo Scientific), as previously described (Lowry, 1972 (link)).

The semi-porous membrane was fabricated by curing the PEGDM resin, sandwiched between glass and a 100 µm thickness silicon mold, with a 365 nm UV lamp (Lightningcure LCS, Hamamatsu Photonics, Hamamatsu City, Japan) for 60 s at an intensity of 310 mW/cm² (Figure A3A). The resulting membrane was glued using an acrylate based instant adhesive (Aron Alpha^®, Tokyo, Japan) to the base of a 12-well insert (Falcon #353180) after removing the existing PET membrane from it. For the diffusion experiment, each insert was loaded with 800 µL FITC-conjugated dextran of either 40 kDa, 150 kDa, or FITC-IgG each of 100 μg/mL concentration, and the well was filled with 2 mL PBS- (Figure A3B). Here, dextran of different molecular weights was used as an evaluation of molecular transport and permeability. The 12-well plate was then put inside a humidified cell culture incubator at 37 °C for 7 days. An end point diffusion measurement was done by taking 200 µL sample from the well and measuring absorption intensity of the sample using a fluorescence plate reader (FluoroscanAscent, Thermo Fisher Scientific, Waltham, MA, USA). Based on a pre-calibrated standard curve, the amount of diffused reagent was determined.

A part of the muscle (~2 mg of d.w.) was homogenized (1:400) in a 0.3 mol/L phosphate BSA buffer adjusted to pH 7.7 and analyzed for the maximal enzyme activity of phosphofructokinase (PFK), hydroxyacyl‐CoA dehydrogenase (HAD), and citrate synthase (CS) using fluorometric methods (Fluoroscan Ascent, Thermo Scientific, Waltham, MA) (Lowry and Passonneau 1972).