Griess reagent kit

The levels of NO in the medium of cultured MSCs were measured as nitrite (NO²⁻) using a Griess reagent kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, 50 µL of culture supernatant was gently mixed with 50 µL of Griess reagent (modified) and incubated in a shaker at room temperature for 15 min. The absorbance at 540 nm was measured in a microplate reader (Cytation5, BioTek).

Nitrite concentration in the lung homogenate was measured (n = 3 per group per time point) using the Griess Reagent kit following manufacturer instructions (Sigma, St. Louis, MO, USA). Absorbance was recorded using a GloMax plate reader (Promega Corporation, Madison, WI, USA).

BV-2 cell culture, treatment and NO production assay. Under an atmosphere of 5% CO₂, BV-2 cells were cultured at 37 °C in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. For the compound screening experiments, the cells were plated in 96-well plates at a density of 5 × 10³ cells/well and 24 h later were treated with compounds 7, 8 or 3-HM at six different concentrations (100, 50, 10, 5, 1 and 0.1 µM). One hour later the cells were treated with 300 ng/mL of LPS (Sigma, USA). After incubation for another 24 h, the culture media was collected for the detection of the NO concentration. The NO accumulation in the medium was determined by measuring the production of nitrite (NO₂⁻) using the Griess Reagent Kit (Sigma, USA).

Lipopolysaccharide (LPS)-induced NO release was evaluated using a Griess reagent kit (Sigma-Aldrich, St. Louis, MO, USA). Briefly, after 24 h on top of the gels, cells were treated with LPS (500 ng/mL; from Escherichia coli O111:B4; CAS Number: 297-473-0, Aldrich) and incubated overnight under permissive conditions. Then, medium from each well was collected, and NO released by RAW264.7 was determined using a Griess test according to the manufacturer’s instructions. Briefly, 75 µL of collected medium from each sample was mixed with 25 µL of Griess reagent and incubated in the dark at RT for 15 min. Then, absorbance at 540 nm was measured by a microplate reader. Treatments were carried out using four replicates. LPS-stimulated cells seeded directly on the well plate were used as a control, taking this result as the 100% NO production (Equation (3)). ANOVA was performed at a significance level of p < 0.05.
$$NOreleased\left[\%\right]=\frac{NOreleased\left(LPS\right)-activatedcells\left(hydrogels\right)}{NOreleased\left(LPS\right)-activatedcells\left(wellplate\right)}\times 100$$

The nitric oxide level in liver, kidney, spleen and brain samples from kefir water treated and untreated groups were measured based on the user’s guideline in the Griess reagent kit (Sigma, USA). The liver, kidney, spleen and brain were mashed individually using plunger and 70 μm cell strainer in phosphate-buffered saline (PBS). The respective supernatants were collected and mixed with deionised water and the provided Griess reagent. The samples were then incubated for 30 min at room temperature. After incubation, the absorbance was measured using a microplate reader (Bio-Tek Instruments, Winooska, VT, USA) at 548 nm. The nitrite concentration in each sample was analysed based on the standard curve of the standard nitric solution.

The lactate concentration was measured with the handheld device DiaSpect Tm (EKF Diagnostics, Germany) according manufacturer’s instructions.
The nitrate concentration was measured using the Szechrome NAS reagent (Polysciences inc., Warrington, USA) according to manufacturer’s instructions. Briefly, the working solution was prepared by mixing 85–86% phosphoric acid and 95–97% sulfuric acid in equimolar amounts. Afterwards 5 g L⁻¹ reagent were added and mixed until the reagent was completely dissolved. Samples were diluted with ddH₂O to the expected sensitivity ratio (1–20 mg L⁻¹) of the reagent, followed by mixing of 100 µL of diluted sample with 1000 µL of the working solution in a cuvette. After 5 min of incubation, the absorption at 570 nm was measured using an Ultrospec2000 pro spectrophotometer.
Nitrite concentration was determined by using the Griess reagent kit (Sigma-Aldrich, st. Louis, USA) according to manufacturer’s instructions. Briefly, 1 g of Griess reagent was mixed with 25 mL ddH₂O. Afterwards, 500 µL of the working solution were mixed with 500 µL of the sample and after 5 min of incubation, measured photometrically at 540 nm.