1 bromo 3 chloropropane bcp

Cell culture materials Medium 254, Medium 254 supplement, fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), and antibiotics were obtained from Gibco (Waltham, MA, USA). Short interfering RNA (siRNA) transfection reagent, Trizol reagent, 1-bromo-3-chloropropane (BCP), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical (St. Louis, MO, USA). ToolScript MMLV RT kit and TOOLS 2× SYBR quantitative real-time polymerase chain reaction (qPCR) Mix was purchased from Biotools (Taipei, Taiwan).

Three independent microglial cultures were treated for 6 h with vehicle (cell culture medium) and 100 ng/mL of LPS (026:B6 Escherichia coli serotype, Sigma Aldrich) with or without 1 ng/mL of recombinant mouse IFNγ (I4777, Sigma Aldrich). Then, total RNA was isolated by lysing pelleted microglial cells from one 75 cm² flask per treatment condition with 1 mL of TriReagent (Sigma Aldrich) and 100 µl of 1-bromo-3-chloropropane (BCP, Sigma Aldrich). The aqueous phase containing total RNA was recovered after centrifuging for 15 min at 12.000 g at 4 °C, mixed with and equal volume of ice-cold 70% Ethanol and loaded onto a PureLink^TM Micro Kit column (Invitrogen). Total RNA was then purified following manufacturer’s instructions. Total RNA was quantified spectrophotometrically using a Nano Drop ND-1000 (Thermo Scientific) and its integrity and quality were assessed with the Bioanalyzer 2100 system (Agilent). Only samples with an RNA integrity number (RIN) greater than 8 were used subsequently for RNAseq and qRT-PCR analyses.

The full-thickness cartilage tissue was removed from subchondral bone and then minced into small pieces and digested with 2 mg/ml pronase (Roche, Basel, Switzerland) for 2 h at 37°C as described in a previous study (Caprez et al., 2018 (link)). Then, cartilage fragments, precooled with liquid nitrogen, were pulverized before transferring into tubes with TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). The pulverized cartilage tissue within TRI was then homogenized with a tissue lyser (Retsch). After centrifugation, the supernatant was transferred into fresh tubes. 1-Bromo-3-chloropropane (BCP, Sigma-Aldrich) was then added into each tube. After 15 min of shaking and 15 min of centrifugation, the aqueous phase containing RNA was transferred into fresh tubes. RNA was then purified with RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA concentration was measured by a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Reverse transcription was conducted with SuperScript Vilo RT Kit (Thermo Fisher Scientific). PCR to assess gene expression was performed with QuantStudio Flex 7.0 instrument and TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA). The primer and probe sequences of tested genes are listed in Table 2. RPLP0 ribosomal RNA was used as endogenous control. Data were analyzed using the 2^−ΔΔCt method.