Hepes

Cell viability assessment was performed on cell monolayers grown to ∼90% confluence in 96-well plates by using the MTS/PMS assay as previously described (Molchanova et al., 2017b (link)). Briefly, the adhered cells were washed with 37°C Hanks’ balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, United States) containing 10 mM HEPES (AppliChem, Darmstadt, Germany), pH 7.4, and exposed for 1 h at 37°C to 100 μL of peptidomimetic dissolved in the medium also used for culturing of each cell line (at concentrations in the range 0–1000 μM). Then the cells were washed twice with 37°C HBSS containing 10 mM HEPES (pH 7.4), and then 100 μL of an MTS/PMS solution, consisting of 240 μg/mL MTS (Promega, Madison, WI, United States) and 2.4 mg/mL PMS (Sigma-Aldrich, Buchs, Switzerland) in HBSS, were added to the cells, which then were incubated for 1 h at 37°C with horizontal shaking (50 rpm) protected from light. A POLARstar OPTIMA plate reader (BMG Labtech, Offenburg, Germany) was used to measure the absorbance at 492 nm. The relative viability was calculated by using 0.2% (w/v) sodium dodecyl sulfate (SDS) as the positive control, while cells exposed to medium without test compound were used as the negative control. Data were obtained in two independent biological replicates performed on separate passages of cells and on separate days with a total number of six replicates.

Small samples (40-100 mg) of liver tissue were rapidly cut on an ice-cold petri dish to prevent oxidation of GSH to GSSG during preparation. Each small sample was immediately placed with a forceps in liquid nitrogen. Samples in the tubes were re-weighed and the weight of the tissue was determined. Ten volumes of ice-cold 5% sulfosalicylic acid (Carl Roth, Karlsruhe, Germany) were added to each tube, the sample was transferred to a tissue grinder and homogenized until evenly suspended. The suspension was added to the same tube and centrifuged at 4°C at 14,000×g for 10 min. The supernatant was transferred to a new tube and equal volume of ice-cold 500 mM HEPES (pH 8) (Gibco, Thermo Fisher Scientific, Schwerte, Germany) were added.
Each sample was diluted 60-fold in ice-cold 250 mM HEPES (pH 7.5) to be in linear detection range for measurement of total and oxidized glutathione using the GSH/GSSG-Glo assay (Promega, Mannheim, Germany). Measurements were performed in duplicate as outlined in the protocol of the manufacturer and as reported elsewhere (Jühlen et al., 2015 (link)).

Adjuvant arthritis FLS were isolated as previously described with modification [40 (link)]. Briefly, fresh synovial tissues were obtained from three adjuvant arthritis rats under sterile conditions each time. All the synovium tissues were minced with fine scissors, incubated in a plastic flask (Corning, New York, NY, USA) and maintained in Dulbecco’s modified Eagle′s medium (DMEM, Gibco, Waltham, MA, USA) supplemented with 10 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, pH 7.2, Promega, Madison, WI, USA), 15% fetal calf serum (FCS, TBD, Tianjin, China), 100 U/mL penicillin and streptomycin (Gibco, Gaithersburg, MD, USA) 50 mM mercaptoethanol in a humidified 5% CO₂-containing atmosphere at 37 °C for 7 days. After removal of the synovial pieces, the adherent cells were cultured in the same medium. At 70–80% confluence nonadherent cells were removed and adherent cells were trypsinized, split at a 1:3 ratio and recultured in the same medium. The synoviocytes were used in experiments from passages 3. After three passages, most of the cultured synoviocytes comprised a homogeneous population of FLS.

The cell line HT-29 was purchased from ATCC (Manassas, VA, USA). The HT-29 cell line was cultivated in DMEM (ATCC) supplemented with 10% FBS (Biowest, Kansas City, MO, USA) and 10 mM HEPES (pH = 7.3, Promega Madison, WI, USA). The conditions of incubation were 37 °C, 4% CO₂, and 95% RH. Assays were carried out in 12-well microtiter plates (MTP) or single vials. Cover slides were placed on them when necessary, and 1 mL of DMEM with 50 µg/mL of gentamicin was then added. A total of 4 × 10⁴ cells were inoculated for 24 h to let them attach.

Regeneration following amputation was quantified at various timepoints, with a different batch of fish used for each respective timepoint. Zebrafish were euthanized in 50 μg/mL benzocaine and fully mineralized bone matrix was visualized using Alizarin red, and newly depositing bone matrix was visualized using calcein green [20 (link)]. Regenerating caudal fins were double-stained with Alizarin red (74 μM Alizarin red S, 5 mM HEPES (Promega, USA) and calcein (40 μM calcein green) made up in 1× Danieau, as previously described [63 (link)]. The region of amputation was imaged under UV excitation through the GFP and RFP filter for calcein and Alizarin red visualization, respectively.