Basic fgf

Cells were maintained in 100 mm culture dishes coated with collagen type I (100 µg/mL). Cells were cultured in endothelial cell basal medium-2 (EBM-2, Lonza, Cologne, Germany) supplemented with 5% fetal calf serum (FCS, PAA Laboratories, Cölbe, Germany), 1% penicillin (100 U/mL), streptomycin (100 µg/mL) (Invitrogen, Karlsruhe, Germany), 5 µg/mL ascorbic acid (Sigma-Aldrich; Munich, Germany), 1% lipid concentrate (Invitrogen), 10 mM HEPES (Invitrogen) and 1 ng/mL basic FGF (Sigma-Aldrich). In addition, 1.4 µM hydrocortisone (Sigma-Aldrich) was included in the medium to reinforce BBB properties [8 (link)]. For induction of Cldn5-YFP expression in Cldn5-YFP-transduced hCMEC/D3 cells, 1 µg/mL doxycycline (Biochrom, Berlin, Germany) was added to the medium during cultivation and the experiments. For monitoring of transendothelial electrical resistance (TEER) cells were seeded in a Transwell^® system (6 well format, polyester membrane, 4.67 cm² growth area, 0.4 µm pore size; Corning Costar Corporation, Cambridge, MA, USA, #3401) at a density of 5 × 10⁴ cells/cm².

Cells were seeded at a density of 1000 cells per well in nonadherent 24‐well culture plates coated with a 10% polyHEMA (Sigma‐Aldrich, St. louis, MO, USA) solution in absolute ethanol and then dried overnight. After seeding, cells were incubated in a serum‐free DMEM‐F12 medium supplemented with 200 ng/mL EGF (Sigma‐Aldrich), 20 ng/mL basic FGF (Sigma‐Aldrich), and B‐27 supplement (Invitrogen). After 5 days of incubation, the number of spheroids in each well was counted under a light microscope (Zeiss, Zena, Germany).

Satellite cells (SCs) were isolated by fluorescence-activated cell sorting (FACS) [6 (link)]. Hindlimb muscles of 8-week-old mice were minced and digested with 0.3% type II collagenase (Sigma) for 1.5 h. The cell suspension was filtered through a 70 μm cell strainer (biosharp), and then centrifuged at 600 g for 5 min. The mononuclear cells were suspended in PBS with 3% FBS for fluorescence staining. Cells were sorted and analyzed by flow cytometry. Markers for SCs isolation were CD31⁻, CD45⁻, CD11b⁻, Sca1⁻, CD34⁺, and Integrin α7 ⁺. Purified SCs were cultured in 24-well plates in Dulbecco’s Modification of Eagle’s Medium (DMEM, Corning) with 20% FBS (Gibco), basic FGF (10 ng/ml, Sigma) and 1% penicillin/streptomycin. Furthermore, when reaching confluence, proliferating cells were incubated with DMEM supplemented with 2% horse serum (Gibco) (differentiation medium, DM) to induce differentiation.
C2C12 cells were purchased from the American Type Culture Collection (ATCC), cultured in DMEM with 10% (v/v) FBS (growth medium, GM). Cells were evenly planked in 6-well plates or 24-well plates with the same cell density, three of which were randomly allocated to control or experimental groups. To induce differentiation, cells were switched into DM after reaching 100% confluence. All cells were cultured in a 37 °C incubator with 5% CO₂.

Culture media α-MEM (α-minimum essential medium), DMEM (Dulbecco’s Modified Eagle’s medium), Medium 199, foetal bovine serum, horse serum, Mitosox, prolong diamond antifade mountant, and mitotracker dyes were all purchased from Thermo Fisher Scientific (UK). α-MEM media-lacking glucose was purchased from PAN Biotech, UK, Mitoquinone (Mito Q) was obtained from Cambridge biosciences, UK). BI605906 was a generous gift from Professor Sir Philip Cohen (MRC Protein Phosphorylation Unit, University of Dundee), but also purchased from Tocris (Bristol, UK), MitoSpy™ Green FM was from BioLegends, UK and MitoPYI, Mitotempo, hepatocyte growth factor, dexamethasone, basic FGF, gelatine, vitamin B12, retinoic acid, VAS2870, palmitate, oligomycin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), rotenone, antimycin-A, hygromycin B, apo-transferrin human, SYBR^® Green JumpStart Taq Ready Mix, and Polyberen were all purchased Sigma-Aldrich, UK).

A total of 1000 CC cells were plated in ultralow attachment plates. The cells were cultured for 10 days in DMEM/F12 medium (Invitrogen, Shanghai, China) supplemented with 4 mg/mL insulin (Sigma, Shanghai, China), B27 (1:50, GIBCO, Shanghai, China), 20 ng/mL EGF (Sigma, Shanghai, China) and 20 ng/mL basic FGF (Sigma, Shanghai, China). For the serial passaging of primary spheres, these were collected, dissociated with trypsin, resuspended in DMEM/F12 medium with the above supplements, and plated to generate secondary spheroids. The number of spheres was counted under the microscope, and the data are expressed as the mean ± SD of triplicate wells within the same experiment.