Complete dmem f12

SIMA9 cells, a widely used immortalized microglial cell line derived from cerebral cortices of mice, were used for cell culture experiments [27 (link)]. This cell line was obtained from the American Type Culture Collection (ATCC^® CRL-3265^TM, Manassas, VA, USA). Cells were cultured in complete DMEM/F12 (Gibco, Carlsbad, CA, USA) with 10% (v/v) FBS (Hyclone Laboratories Inc., Logan, UT, USA), 5% (v/v) horse serum (Gibco, Carlsbad, CA, USA), and 1% (v/v) Penicillin-Streptomycin (Gibco, Carlsbad, CA, USA). Cells were passaged with a subculturing solution containing 1 mM EDTA, 1 mM EGTA, and 1 mg/mL glucose. Human cerebral microvascular endothelial cells (hCMEC/D3) were used in this study. hCMEC/D3 cells have been widely used to develop an in vitro model of the human BBB [28 (link)]. This cell line was purchased from MilliporeSigma (#SCC066, Burlington, MA, USA). hCMEC/D3 cells were cultured on 5–10 µg/cm² of collagen type I (Gibco, Carlsbad, CA, USA) and maintained in Endothelial Cell Growth Medium MV2 (PromoCell, Heidelberg, Germany), 5% (v/v) FBS, and 1% (v/v) Penicillin-Streptomycin. hCMEC/D3 cells were subcultured with 0.05% (v/v) Trypsin. Both cell lines were incubated with humidified 5% CO₂ at 37 °C and were plated overnight (24 h) before being used for experiments.

RAW264.7 cells were routinely cultured in α‐MEM (Gibco, Waltham, MA, USA) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin at 37 °C in a 5% CO₂ condition. Rosiglitazone (Cayman Chemical, Ann Arbor, MI, USA) was added to the medium 30 min before RANKL (R&D Systems, Minneapolis, MN, USA) administration, and cells were maintained for an additional 24 h. For siRNA experiments, complete DMEM/F12 (Gibco) without antibiotics was used as the culture medium.

Mouse primary alveolar epithelial cells were isolated as described previously (49 (link)). Briefly, cardiac perfusion was performed using DPBS (Life Technologies). Subsequently, a cannula was placed in the trachea to infuse, first, dispase (50 U/ml, Roche) for ∼45 seconds to allow the enzyme to distribute throughout the lungs without allowing it to spill back out. Next, 1 ml of 1% low-melt agarose (Sigma Aldrich) was gently infused. Lungs were covered with crushed ice for 2 minutes to allow for the agarose to solidify. Individual intact lung lobes were cut away and additionally kept in a dispase solution (50 U/ml, Roche) for 45 min at room temperature on a rocker at 150 rpm. After 45 minutes digested lungs were decanted in complete DMEM/F-12 (Gibco, Life Technologies) with DNase I (Qiagen) for 10 minutes to avoid clumping of cells. Single cell preparations were serially strained though a 100 μm, 70 μm and 40 μm strainers. Primary epithelial cells (singlets, alive, CD45-, EpCAM+ cells) were isolated with a FACSAria III cell sorter (BD) with a cell purity typically around 96%. The gating strategy is depicted in Supplementary Figure S3B.

IMMUNEPOTENT CRP^© (ICRP), a bovine dialyzable leukocyte extract, was produced by Laboratorio de Inmunología y Virología from Facultad de Ciencias Biológicas as previously described (Franco-Molina et al., 2006[10 (link)]). The product obtained from 1x10⁸ leukocytes is defined as one unit of ICRP. ICRP and Cyclophosphamide (Cryofaxol from Cryopharma; Tlajomulco de Zuñiga, Jalisco, México) were dissolved in complete DMEM-F12 or RPMI (GIBCO by Life Technologies, Grand Island, NY), as suitable. N-acetyl-L-cysteine (NAC) was dissolved in water. QVD.opH (QVD) was dissolved in dimethyl sulfoxide (DMSO). CTX, NAC and QVD (Sigma-Aldrich, St. Louis, MO) were wrapped in foil and stored following the manufacturer's instructions.

Isolation of articular chondrocytes was carried out through several cycles of enzymatic digestion. Briefly, cartilage was mechanically disrupted into small pieces (1 mm³ approximately) using a scalpel, and subsequently subjected to enzymatic breakdown (30 min at 37 °C with constant stirring at 90 rpm) with trypsin without EDTA (GIBCO^®-BRL Life Technologies, Grand Island, NY, USA). Then, digestion continued with at least two cycles of type II collagenase 2 mg/mL at 37 °C with constant stirring at 90 rpm. Cells contained in the supernatant were pelleted via centrifugation at 1800 rpm for 5 min for each digestion cycle. Chondrocytes were suspended in complete DMEM/F12 (GIBCO^®-BRL Life Technologies, Grand Island, NY, USA) culture medium supplemented with 10% fetal bovine serum (FBS, GIBCO^®-BRL Life Technologies, Grand Island, NY, USA) and gentamicin (0.05 mg/mL, Laboratorios Química SON’S, Puebla, Mexico), and transferred to 75 cm² culture flasks at 37 °C in a 5% CO₂ environment and relative humidity of 100%. Cells with less than three passages were used.