Penicillin streptomycin pen strep

IGRA was performed as previously described (21 (link)); 1.5 ml aliquots of heparinized blood diluted 1:1 in RPMI medium (Fisher, Hampton, USA) supplemented with 1% sodium heparin (Roche, Basilea, Switzerland) and 1% penicillin/streptomycin (pen/strep) (Fisher, Hampton, USA) was stimulated with a final concentration of 30 μg/ml bovine and avium tuberculins (Spanish PPD-B and PPD-A, respectively, CZ Vaccines, Porriño, Spain), with pokeweed mitogen at a final concentration of 5 μg/ml (Sigma-Aldrich, St. Louis, USA), with CFP-10/ESAT-6 (Lionex, Braunschweig, Germany) at a final concentration of 5 μg/ml protein cocktail or without antigen (RPMI+Pen/strep), and kept at 37°C with 5% CO₂ for a minimum of 16 h. Post-incubation, supernatants were collected in duplicate aliquots (250 μl) and stored at −80°C. IFN-γ levels were assessed by sandwich ELISA using anti-badger IFN-γ capture monoclonal antibody 10H6-C1 and biotinylated monoclonal antibody 11B9 (Animal and Plant Health Agency, UK). The cut-off point of 0.044 (using combined PPD-B and PPD-A values) was used to identify positivity to M. bovis infection.

The C2C12 cells were grown with Dulbecco’s Modified Eagle’s Medium (DMEM, Hy-clone, Pittsburgh, PA, USA) supplemented with 10% fetal bovine serum (FBS; Gemini Bioproducts, West Sacramento, CA), 1% penicillin/streptomycin (pen/strep; Fisher Scientific, Pittsburg, PA, USA), and 1% antibiotic/antimycotic (anti/anti; Gemini Bioproducts, West Sacramento, CA, USA) in T25 flasks until 70–75% confluency. Cells were then split into T75 flasks and when confluent, they were plated in 24-well plates at a cell density of 1 × 10⁵ cells/mL.

Dulbecco’s modified Eagle medium nutrient mixture/F-12 medium (DMEM-F12) containing L-glutamine, penicillin/streptomycin solution, and Corning regular fetal bovine serum (FBS) were purchased from (Fisher Scientific, Grand Island, NY, USA), Penicillin/Streptomycin (Pen-Strep) (Fisher Scientific, Grand Island, NY, USA), and Amphotericin-B were also obtained from (Fisher Scientific, Grand Island, NY, USA). Climbazole (Tokyo Chemical Industry, Tokyo, Japan) and Heparin sodium salt (EMD Millipore Corporation, Burlington, MA, USA) were the inhibitors used in this study. Unless otherwise indicated, all other drugs were purchased from commercial sources. A549 cells were used in this study. A549 cells were obtained from (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in DMEM-F12 (Fisher Scientific, Grand Island, NY, USA) supplemented with 10% FBS, 1% Pen-Strep, and 0.2% Amphotericin-B (0.5 μg/mL). For virus infection, DMEM exosome-free media was prepared with exosome-depleted FBS using DMEM/F12 medium containing L-glutamine supplemented with 2% exosome-free Corning FBS, 1% Pen-Strep, and 0.2% Amphotericin-B (0.5 μg/mL) (Fisher Scientific, Grand Island, NY, USA). Cells were cultured at 37°C in a humidified atmosphere supplemented with 5% CO2.

Trabecular and cortical bone were extracted from the femoral heads and placed into a 50 mL conical tube containing 10 mL of Hanks′ Balanced Salt Solution (HBSS). Followed by bone removal, the bone marrow of the samples was washed with additional HBSS to collect bone marrow cells. After the cells settled for 2−3 min, the solution was filtered through a 70 µm cell filter into a separate 50 mL falcon tube containing 5 mL of alpha modified Minimum Essential Medium Eagle (α-MEM; Caisson Labs, Smithfield, UT, USA, Cat# MEL08-500ML), supplemented with 10% fetal bovine serum (FBS; Gemini Bioproducts, West Sacramento, CA ,USA), 1% penicillin/streptomycin (pen/strep; Fisher Scientific, Pittsburg, PA, USA), and 1% antibiotic/antimycotic (anti/anti; Gemini Bioproducts, West Sacramento, CA, USA). The filtered solution was centrifuged at 1800 revolutions per minute (RPM) for 9 min at 4 °C. The cell pellet was resuspended in α-MEM and plated into 12-well or 24-well plates at a cell density of 1 × 10⁵ cells/mL (Figure 2).

Fibroblasts were isolated from the human synovial tissue samples described above. A portion of the tissue fragments were transferred to a 15 ml conical centrifuge tube containing 5 ml of collagenase D (Sigma Aldrich 11088858001) at a concentration of 1 mg/ml (dissolved in Hank’s balanced salt solution (HBSS) [Sigma Aldrich 55037C] + 1% Penicillin/Streptomycin (Pen/Strep) [Fisher Scientific 15140122]). The tube was kept in a 37°C bead bath for 1 hour and was shaken vigorously every 5 minutes to release cells. Large tissue fragments were removed using sterile forceps and discarded. Remaining liquid was centrifuged at 1100 rpm for 10 minutes at room temperature. Supernatant was discarded and cell pellet was resuspended in 5 ml of enriched human fibroblast medium (High glucose DMEM [Sigma Aldrich D5671] + 20% fetal bovine serum (FBS) [BioWest S1690] + 1% Pen/Strep + 1% glutamine [Fisher Scientific 35050061] + 1% non-essential amino acids (NEAA) [Fisher Scientific 11140050] + 5 ng/ml recombinant human FGF-basic [BioLegend 792504]). Cells were then transferred to a T25 tissue culture flask and placed in a 37°C incubator with 5% CO2. Cell culture medium was replaced every 3–4 days, and cells were passaged at ~ 90% confluency. Primary fibroblasts were frozen at passage 4 and stored in liquid nitrogen.

MDCK32 (link), 21D130 (link)32 (link), 21D1^−MMP1, MEF (ATCC) and HUVECS (ATCC) were maintained at 37 °C with 10% CO₂ in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Carlsbad, CA, USA) containing 10% (v/v) Fetal Calf Serum (Life Technologies), 1% (v/v) Penicillin Streptomycin (Pen/Strep) (Life Technologies).