Colld ro

For murine chondrocyte experiments, chondrocytes were isolated from sterna of newborn pups (C57BL/6 J) age P1-P3 without consideration for sex. Cells were isolated by sequential digestion with pronase (2 mg/mL, PRON-RO, Roche) at 37°, followed by collagenase D (3 mg/mL, COLLD-RO, Roche) two times at 37°, and cultured in DMEM (Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (15140122, ThermoFisher, Waltham, MA, USA) and plated in tissue culture plates. For glutamine deprivation conditions, media was changed to high glucose DMEM containing glutamine or devoid of glutamine (Life Technologies, Carlsbad, CA, USA). For experiments, cells are treated with recombinant mouse IL-1β (211-11B, Peprotech, Cranbury, NJ, USA) at 10 ng/mL, CB-839 (S7655, Selleck, Chemicals, Houston, TX, USA), rapamycin (HY-10219, MedChem Express, Monmouth Junction, NJ, USA), ammonium chloride (A9434, Sigma-Aldrich, USA), L-asparagine (A0884, Sigma-Aldrich, St. Louis, MO, USA), or L-glutamatic acid (G1626, Sigma-Aldrich, St. Louis, MO, USA).

All reagents were purchased from Sigma-Aldrich (UK) or Life Technologies (UK) unless otherwise stated. Aliquots of Dithiothreitol (DTT) (Life Technologies) were pre-prepared in distilled H₂O and stored at −20^°C. The wash solution (mucolytic solution) used in the initial steps of enterocyte isolation contained 1% penicillin/streptomycin and an aliquot of DTT dissolved in Hanks Buffered Saline Solution (HBSS) with MgCl₂ and CaCl₂ which gave a final concentration of 1 mM. Fresh wash solutions were made for each isolation due to DTT's reduced activity at room temperature as previously observed by Goodyear et al. (2014) (link). Solution formulations can vary between manufacturers and significantly impact the type and success of primary cultures of RT tissue as previously observed by Ganassin and Bols (1996) (link) in cultures of RT spleen. As such, trypsin (25050014), versene (15040033) and trypsin/EDTA (25300054) were purchased from Life Technologies. The enzyme digestion solution contained 0.1% collagenase D (COLLD-RO, Roche) and dispase II (D4694, Sigma-Aldrich) (equivalent to 1 mg/ml) with DNase (0.1 mg/ml) and 1% FBS/BSA. This solution is pre-warmed to 37°C prior to enterocyte isolation as is routine in many primary culture protocols.

Primary chondrocytes were harvested from sterna and ribs of P1-P3 mice using sequential digestion with pronase (2 mg·mL⁻¹, PRON-RO, Roche) at 37 degrees, collagenase D (3 mg·mL⁻¹, COLLD-RO, Roche) two times at 37 degrees, and cultured in DMEM (Life Technologies, USA) containing 10% FBS and 1% penicillin/streptomycin (Thermo Fisher). Primary chondrocytes are not passaged and experiments are completed within 5 days of isolation from mice. Primary macrophages (osteoclast precursors) were harvested from long bones of WT mice and cultured in M-CSF overnight before being added to chondrocyte cultures. Cells were incubated with recombinant IL-1β (10 ng·mL⁻¹), IKK2 inhibitor SC-514 (10 μmol·L⁻¹), BP (1 mg·mL⁻¹). Plat-E cells were used to generate retroviral particles.