Dulbecco s modified eagle medium dmem

The sterilization of CNT forests started with soaking the specimens in 70 vol% ethanol, followed by overnight ultraviolet (UV) light exposure with 40 µW/cm² intensity at a distance of 724 mm. Then, the specimens were washed with PBS-Pen/Strep three times for 30 min each. The gelatin solution (1 wt%) was prepared by dissolving gelatin in PBS-Pen/Strep solution at 50 °C for 60 min. The gelatin solution was filtered using a 0.22 µm filter (Thermofisher, US). CNT forests were placed in the gelatin solution and incubated at 37 °C and 5% CO₂ for 2 h. The gelatin solution was then removed, and the CNT forests were washed with PBS-PenStrep twice for 5 min each. The scaffolds were then placed in a cell culture medium relevant to the specific cell used (Dulbecco's Modified Eagle Medium (DMEM) (ATCC, US) for 3T3 and DMEM for primary cell isolation (with high glucose for cell isolation) (Thermofisher, US) for CM) and incubated at 37 °C and 5% CO₂ for at least 1 h before cell seeding.

Primary human dermal fibroblasts (ATCC, Manassas, USA) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. All cell culture materials were purchased from Thermo Fisher Scientific, Dreieich, Germany.
For cell culture, 3 × 10⁴ fibroblasts were seeded onto reconstituted 3D collagen matrices. Cells were cultured for 3 days under standard cell culture conditions (37 °C with 5% CO₂ and 95% humidity). For differentiation of fibroblasts into myofibroblasts as a control, cells were cultured in DMEM supplemented with 10 ng mL⁻¹ TGF‐β1 (Biolegend, San Diego, USA) for 3 days under standard cell culture conditions, as previously described.^[40]

Cell lines for measuring anti-inflammatory activity human monocyte (THP-1) and murine macrophages (RAW 264.7) were used. All were provided by the American Type Culture Collection (ATCC, USA). Additionally, Rosewell Park Memorial Institute (RPMI) 1640, Dulbecco’s Modified Eagle Medium (DMEM) were purchased from ATCC. Fetal bovine serum (FBS), penicillin-streptomycin, sodium pyruvate, and glutamine were purchased from Biological Industries (Beit HaEmek, Israel). 2-Mercaptoethanol was purchased from BIO-RAD and ELISA kits were purchased from PeproTech. LPS was purchased from Sigma-Aldrich (Sigma-Aldrich, Israel).

All chemicals (Hexane, methanol –LC-MS (≥99.9%), water, and ethanol absolute proof (≥99.5%) were all high-performance liquid chromatography (HPLC) grade from Sigma Aldrich, MO, USA. Capsaicin and dihydroCapsaicin standards were obtained from Santa Cruz Biotechnology Inc., Dallas, TX, USA. Dulbecco’s Modified Eagle Medium (DMEM) and FBS were purchased from ATCC (Manassa, CO, USA). The concentrations of the Capsaicin and dihydroCapsaicin standards were evaluated using a stock solution of 6 mg/mL Capsaicin and a stock solution of 5 mg/mL dihydroCapsaicin. The standards were dissolved completely in a 10:1 Hexane-ethanol solution [31 (link)].

Sca1⁺‐VPCs were isolated from the outgrowth of adventitial tissues of mouse arterial vessels, as previously described.12, 13 Briefly, the arterial vessels were harvested from C57BL/6J mice (Charles River, Margate, Kent, UK) or Hd7‐7sFLAG transgenic mice and cut into 2‐mm rings after the removal of the intima and media; the pieces were placed in gelatin‐coated flasks and incubated at 37°C in a humidified incubator supplemented with 5% CO₂ for 6 hours. Stem cell culture medium ([Dulbecco's modified Eagle medium (DMEM); ATCC, Rockville, Maryland] supplemented with 10 ng/mL recombinant human leukemia inhibitory factor [Chemicon, Temecula, California], 10% fetal bovine serum [FBS, ATCC], 0.1 mmol/L 2‐mercaptoethanol, 100 U/mL penicillin, and 100 U/mL streptomycin) was added and refreshed every other day until the cells reached 80% confluence. The cells were expanded and subjected to Sca‐1⁺ cell purification using anti‐Sca‐1 immunomagnetic microbeads Miltenyi Biotec (Bergisch Gladbach, Germany). The purity of isolated Sca‐1⁺ cells was confirmed to around 85% using flow cytometry.12, 13, 14 The Sca‐1⁺‐VPCs were maintained in stem cell culture medium and split every other day. Cells passaged up to 30 times were used in this study, and Sca1‐selection was performed every five passages.

The 3T3-L1 mouse embryo fibroblasts and Dulbecco’s modified Eagle medium (DMEM) were obtained from ATCC (Rockefeller, Maryland, USA). Bovine serum and fetal Bovine serum (FBS) were purchased from GIBCO (Invitrogen, CA, USA). The primary antibodies (anti-PPARγ and anti-Twist 1) were obtained from Abcam. The HRP-conjugated secondary antibody and the anti-β-actin primary antibody were purchased from ZSGB-BIO. The lentiviral vectors pGLV3/H1/GFP + Puro (LV3), LV4-EF1a-GFP/Puro (LV5), recombinant vectors LV3/Twist 1 shRNA and LV5/Twist 1 complementary DNA (cDNA) were all acquired from GenePharma (Shanghai, China). NE-PER™ Nuclear and Cytoplasmic Extraction Kit was purchased from Pierce (Thermo Scientific). SYBR^® Green Real-time PCR Master Mix (QPK-201 T) was purchased from TOYOBO. T0070907 and pioglitazone were both from Cayman Chemical (MI, USA). Insulin, isobutylmethylxanthine, cycloheximide (CHX), leupeptin, pepstatin A (L/P), proteasomes inhibitors (MG132) and the other reagents in this study were obtained from Sigma (Merck, German).