Recombinant mouse hgf

Single myofibers were obtained by collagenase digestion of EDL and peroneus muscles, as previously described (Hawke et al. 2003 (link)). Briefly, following collagenase digestion, muscles were triturated with plastic Pasteur pipettes and moved to cell culture dishes with a glass Pasteur pipette. Floating cultures were achieved by coating dishes with 10% normal horse serum prior to addition of plating media (10% normal horse serum, 0.5% chick embryo extract [MP Biomedicals] in low-glucose [1 g/L] Dulbecco’s modified Eagle’s medium [DMEM; Invitrogen]). Single myofibers were incubated in one of three conditions: basal (plating media alone), Recombinant Mouse HGF (R&D, 10 ng/mL), or dimethylsphingosine (DMS, 10 μmol/L) for 45 min, prior to the addition of BrdU to the medium. Satellite cell activation was determined by performing immunoflu-orescence on single myofibers following a 24-h incubation in 5-bromo-2-deoxyuridine (BrdU; Sigma, 10 μmol/L), as previously described (Nissar et al. 2012 ). The number of BrdU-positive SCs per myofiber were counted on at least 12 myofibers per treatment group as a measure of the functionality of SCs becoming activated by HGF.

For colony-formation assays, isolated luminal cells were plated on irradiated 3T3 cell feeders in 24-well plates at a density of 500 cells per well and cultured in DMEM/F12 medium supplemented with 10% FBS, 5 μg/mL insulin (Sigma-Aldrich), 10 ng/mL EGF (Invitrogen, Life Technologies), and 100 ng/ml cholera toxin (ICN Biochemicals) for 7–8 days, as previously described [8 (link), 23 (link)].
For mammosphere-formation assays, 5000 isolated luminal cells were seeded on ultralow-adherence 24-well plates (Corning) in DMEM/F12 medium supplemented with 2% B27 (Stem Cell Technologies), 20 ng/mL EGF, 20 ng/mL bFGF (GIBCO, Life Technologies), 4 μg/mL heparin (Sigma-Aldrich), 10 μg/mL insulin, and 2% growth-factor-reduced Matrigel (BD Biosciences) for 10–12 days. For second-generation sphere assays, mammospheres were dissociated with 0.05% trypsin (Gibco, Life technologies) and reseeded as described above. When specified, cells were treated with 25–50 ng/ml recombinant mouse HGF (R&D Systems Europe) every 2 days, as described elsewhere [8 (link)]. ImageJ software (NIH) was used to count colonies and mammospheres and quantify their size in pixels.

Bone marrow from murine tibiae was flushed with PBS and cultured in αMEM media with 10% FCS (37°C, l 5% CO₂) for 24 h to allow stromal cells to attach. After 24 h the cells were harvested and seeded onto sterile dentine discs (sterilised in an ultrasound bath and pre-soaked in αMEM media) in 96 well plates at 0.5 x 10⁶ cells per well in 100 μl αMEM media with 10% FCS, recombinant mouse M-CSF (150 μg/ml, R and D Systems, Oxford, UK) and recombinant mouse RANK-L (30 μg/ml, R and D Systems). After 24 h, the cells were washed (4 times with media excluding M-CSF and RANK-L) and then 200 μl fresh media containing M-CSF/RANK-L added to each well. After a further 48 h, the used media was removed and fresh media added (containing M-CSF/RANK-L) with either DMSO (N = 2), 1 μM ARQ-197 (N = 2) or 1 μM ARQ-197 with 50 ng recombinant mouse HGF (R and D systems) (N = 2). The cells were treated 3 times a week for 2 weeks. After 2 weeks the discs were fixed in 10% buffered formalin overnight and stained for tartrate-resistant acid phosphatase (TRAP) [35 ]. Images were taken under light microscopy.