Na2hpo4

The lysis/binding buffer consisted of 15.76 g Tris‐HCl (AppliChem GmbH, ≥99%), 21.197 g LiCl (Carl Roth GmbH & Co. KG, pure ≥98.5%) and 10 g SDS (Carl Roth GmbH & Co. KG, ultrapure) in 1 L deionized water. pH of 7.5 was adjusted with 1 M NaOH (Carl Roth GmbH & Co. KG, 1 N measured solution). Note: The substances DTT and EDTA, which are often used in lysis buffers, were omitted because their use in interaction with Dynabeads is not recommended [59 ].
PBS buffer was composed of 0.294 g NaH₂PO₄ * 2H₂O (Merck KGaA, ≥99%), 1.44 g Na₂HPO₄ * 2 H₂O (Carl Roth GmbH & Co. KG, ≥98%) and 8.78 g NaCl (AnalaR Normapur, >99.5%) in 1 L deionized water. Note: Commercially available oligo(dT)‐functionalized 2.8 μm Dynabeads for mRNA purification are supplied in this buffer [60 ].

Ab-HRP was obtained from the ELISA kit produced by Institut Pourquier (Montpellier, France). Hydrogen peroxide, K₄[Fe(CN)₆] K₃[Fe(CN)₆] was delivered by Carl Roth GmbH (Berlin, Germany). In addition, 25% of glutaraldehyde solution was delivered by Fluka Chemie GmbH (Buchs, Switzerland). Moreover, 98.5% of ethanol solution from “Vilniaus degtinė” (Vilnius, Lithuania) was used for electrodes and plastic cells cleaning.
The supporting electrolyte for the electrochemical experiments was 0.1 M acetate- phosphate buffer (A-PBS), pH 6.5, which was prepared using NaH₂PO₄ from Fluka Chemie GmbH (Bucharest, Romain), Na₂HPO₄ from Carl Roth GmbH (Berlin, Germany), KCl from Scharlau (Barcelona, Spain), and CH₃COONa from Merk (Tokyo, Japan). The pH was adjusted with CH₃COOH from Merk (Darmstadt, Germany) or NaOH from Merk (Steinheimm, Germany).

CO₂ pure grade (99.95%, Air Liquide, Lisbon, Portugal) and EtOH (96%) were used for high pressure extraction experiments. Solvents used for conventional extractions purification steps and chromatographic analysis included ethyl acetate (99.98%, Fisher scientific U.K. Limited, Loughborough, UK), methanol (99.8%, Fisher scientific U.K. Limited, Loughborough, UK), dichloromethane (Honeywell Riedel-de Haën, Germany), distilled water, and ultrapure water purified with a Milli-Q water purification system (Merck Millipore, Billerica, MA, USA). For the inflammation assays, the inflammatory stimulus was carried out by the usage of MnCl₂ (Merck, Darmstadt, Germany), and the anti-inflammatory positive control was carried by FK506 (Cayman Chemicals, Ann Arbor, MI, USA). Cellular lysis was preformed using Yeast Protein Extraction Reagent (Y-PER; ThermoFisher Scientific, Scientific, Rockford, IL, USA). To quantify the expression of reporter gene lacZ, we employed a solution buffer composed of Na₂HPO₄ (ROTH, Karlsruhe, Germany), NaH₂PO₄·H₂O (Merck, Buchs, Switzerland), KCl (Panreac, Barcelona, Spain), and MgSO₄·7H₂O (Merck, Buchs, Switzerland), containing o-nitrophenyl β-D-galactopyranoside (ONPG; Sigma–Aldrich^®–Poole, Dorset, UK). Nuclear staining was carried out using nuclear dye Hoechst 33342 (Sigma, Buchs, Switzerland).

Culture medium (CM) was prepared using RPMI 1640 supplemented with 100 U/mL of penicillin, 100 μg/mg streptomycin (both from Sigma, Steinheim, Germany), and 2 mM L-glutamine (Biochrom, Berlin, Germany). Concanavalin A (ConA) (Sigma) was diluted in CM to a concentration of 600 μg/mL and stored at −70°C. Antibodies were purchased from Becton Dickinson (BD, Heidelberg, Germany). Phosphate buffered saline (PBS) was made by dissolving 7.013 g NaCl, 0.2 g KCl, 1.513 g Na₂HPO4, and 0.2 g KH₂PO4 (all purchased from Roth) in 1 liter distilled water and by adjusting the pH to 7.4. Red blood cell (RBC) lysing solution was purchased from Becton Dickinson and diluted 1 : 10 in distilled water before use. Washing buffer was obtained from BD Biosciences (San Diego, USA). Formaldehyde solution and absolute methanol was purchased from Merck (Darmstadt, Germany).

Cell culture flasks and 96-well plates were purchased from Sarstedt (Nürnbrecht, Germany). Cell culture media (Dulbecco’s Minimal Essential Medium (DMEM) and Eagle’s Minimal Essential Medium (EMEM) without phenol red) and supplements (fetal bovine serum (FBS), charcoal–dextran stripped FBS (CD-FBS), blasticidin S HCl and geneticin (G418)) were produced from Gibco and obtained from Thermo Fisher Scientific (Waltham/MA, USA). ZEN, α-ZEL, E2, (Z)-4-hydroxytamoxifen (4-OH-TAM) and sulforhodamine B (SRB) were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). DAI, EQ, and GEN were obtained from Extrasynthese (Genay Cedex, France) whereas dimethly sulfoxide (DMSO), NaCl, KCl, Na₂HPO₄, Na₂HPO₄ * 2 H₂O, and KH₂PO₄ were purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). The CellTiter-Blue^® Cell Viability Assay Kit and ONE-Glo™ EX luciferase assay system were obtained from Promega Corporation (Madison/WI, USA).

To calculate DNA content, pieces of tendon tissue recovered from the centre of each sample were subjected to papain digestion. For this purpose, 200 mg of each sample were minced into 1 mm³ pieces and washed in PBS. Subsequently, the samples were incubated in 800 μl papain digestion buffer [2 mM n-acetyl-l-cysteine (Sigma Aldrich), 2 mM EDTA (Carl Roth), 50 mM Na₂HPO₄ (Carl Roth)] and 20 μl papain solution (10 mg/ml) (Sigma Aldrich) for 24 h at 60 °C. Storage of digested tissue samples before further analysis took place at -20 °C.
DNA content was measured using the Quant-IT™ PicoGreen® dsDNA assay kit (Thermo Fisher Scientific). To achieve this, digested tissue samples (10-fold dilution for internal controls) were pipetted in 96-well plates and an equal volume of PicoGreen® reagent working solution was added. Subsequently, the samples were incubated for 5 min at room temperature, protected from light. Fluorescence (excitation wavelength of 480 nm) was measured by a microplate reader (SynergyTM H1 Hybrid Multi-Mode Microplate Reader, Gen5TM Software, BioTek® Instruments, Inc.). DNA content was determined by the use of standard curves prepared from DNA standards measured on the same plates.
Results from DNA measurement were again normalized to the respective controls and are given as percentages relative to controls.

TTC and Evan’s blue staining were used to quantify the myocardial repAMI size as described previously [30 (link),31 (link)]. Hearts were removed and flushed blood-free with PBS (Sigma-Aldrich, St. Louis, MO, USA) via cannulation of the ascending aorta. The LCA was then re-occluded by suture followed by injection of 1 mL 1% Evan’s blue dye (Sigma-Aldrich, St. Louis, MO, USA) to visualize the area at risk. Then, 1–2 mm transversal cross-sections of the myocardium were created and incubated in 1% TTC in 0.0774 M Na₂HPO₄ (Roth, Karlsruhe, Germany) and 0.0226 M NaH₂PO₄ (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 5 min. Images were obtained with an M80 microscope with an IC80 HD camera (Leica, Wetzlar, Germany) and analyzed by a blinded investigator using ImageJ software.