gments were digested with 2% collagenase II for 60 min at 37uC. Digested cells were discarded and fragments were incubated again with 0.05% trypsin for 15 min at 37uC with gentle agitation. After the incubation, isolated cells were collected Materials and Methods Magic-F1 Factor engineering and purification Magic-F1 is an engineered factor containing two HGF NK2 domains joint by a linker. The exact amino acidic sequence of Magic-F1 corresponds to: residues 1285 of human HGF; a linker with the sequence 3; residues 30285 of human HGF; a poly-histidine tag with the sequence Inducing Muscular Hypertrophy and fragments were incubated again until the whole tissue was digested. Isolated cells were pooled, centrifuged and resuspended in DMEM supplements with 20% pre-screened FCS, 1% gentamycin, and plated onto collagen coated dishes at a density of 104 cells6cm2. Contamination by non-myogenic cell was reduced by pre-plating the cell suspension onto plastic dishes where fibroblasts tend to adhere more rapidly. Differentiation was induced shifting the medium to DMEM supplemented with 2% horse serum. Cell morphology was examined daily with a phase-contrast microscope connected to an image analyzer. Cells were Astragalus polysaccharide biological activity trypsinized daily and counted on a hemocytometer. Cell viability was determined by trypan blue dye exclusion assay. Cell cytotoxicity was performed using an XTTbased in vitro toxicology assay kit according to manufacturer’s protocol. Incubation medium was collected after 3 hours and read spectrophotometrically at a wavelength of 450 nm. Background signals, obtained from plates without cells, were subtracted from sample readings. Apoptosis was quantified using an ApopTag Fluorescein In situ Apoptosis detection kit according to the manufacturer’s protocol. Cell differentiation was carried out for 8 days. Cells were grown on 6 cm Petri dishes until sub-confluent, washed with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes and then permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. After incubation with PBS 15976016 containing 10% normal serum, samples were incubated overnight at 25137254 4uC with antiGFP at 1:200 dilution, anti myosin heavy chain antibody at 1:2 dilution. After incubation, cells were washed three times in PBS and incubated with the appropriate FITC- or TRITCconjugated secondary antibodies for 1 hour at room temperature. After 
washing in PBS, cells were analyzed under a fluorescent microscope and photographed. As a control for the immunofluorescence method, we omitted the primary antibody and no staining was detected under these conditions. Cell nuclei were counterstained with DAPI. the leg skin was ensured by shaving each leg and applying a conductive gel. Square-wave electric pulses were generated by a digital Stimulator. Muscular regeneration analysis Acute skeletal muscle damage was induced in male and female MLC1F/Magic-F1transgenic mice and control mice by i.m. injection of 10 nM cardiotoxin in physiologic solution. Control mice were injected with physiologic solution alone. At 3, 7, and 14 days after drug injection, mice were sacrified and subjected to histological evaluation and morphometric analysis of tibialis anterior. After excision, muscles were sectioned and processed for immunofluorescence analysis using the primary antibodies listed above. All sections were washed three times in PBS and incubated with 10% donkey serum for 30 min at RT before the addition of the appropriate Alexa 488-, Alexa 594- or Al
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