Applied biosystems power up sybr green pcr master mix

The total RNA from the cultured BMMs was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's recommended protocol. All experiments were conducted only with high-quality RNA (A260/A280 ratio of 1.8–2.0 and A260/A230 ratio of 2.0–2.2). Reverse transcription was performed with 1 μg of RNA and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol. The cDNA was amplified by using Applied Biosystems Power-Up SYBR green PCR master mix (Thermo Fisher Scientific) and quantified by using Quantstudio®5 Real-Time PCR (Thermo Fisher Scientific). Primers were used as previously reported [17 (link)], and the genes were normalized to encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All real-time PCR reactions were performed at least three times, and all data were analyzed by using the 2^−ΔΔCt method [22 (link)].

All reagents for liquid chromatography (LC) and mass spectrometry (MS) analysis were of LC-MS grade and purchased from Merck (Darmstadt, Germany). Trypsin Gold was purchased from Promega (Madison, WI, USA). C18 solid phase extraction columns were purchased from Waters Corporation (Milford, MA, USA). Millipore Protease Inhibitor Cocktail Set I was purchased from Merck (Darmstadt, Germany). Invitrogen SuperScript™ IV First-Strand Synthesis System and Applied Biosystems PowerUp SYBR Green PCR Master Mix were purchased from Thermo Fisher Scientific (Vilnius, Lithuania). Ambion TRIzol Reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA). Invitrogen nucleophosmin monoclonal antibody (NA24), Invitrogen natriuretic peptides A polyclonal antibody, Pierce BCA Protein Assay Kit and Pierce ECL chemiluminescence kit were purchased from Thermo Scientific (Rockford, IL, USA). The moesin polyclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The S100A8/A9 inhibitor (ABR-238901) was a gift from Active Biotech AB (Lund, Sweden).

Primers were chosen using the Primer3 online tool. Primer sets used in this study are shown in Table S1. Total RNA was extracted from C2C12 cells using Trizol reagent (Invitrogen). First stand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Real‐time PCR was performed using Applied Biosystems Power‐Up SYBR green PCR master mix (Thermo Scientific) and detected using Quantstudio®5 Real‐Time PCR (Thermo Scientific). The gene encoding GAPDH was used as an internal standard. All reactions were performed in triplicate, and data were analyzed using the 2^−ΔΔCt method (Livak & Schmittgen, 2001).