The decline in nestin-GFP expression in muscle progenitor cells coupled to its persistence in neural precursor cells indicates the discovery of a new tool to address these clinically relevant problems. Materials and Methods Animals Nestin-GFP transgenic mice were maintained as homozygous for the transgene on the C57BL/6 genetic background [15]. C57BL/6 wild-type mice were used as the control. All mouse colonies were housed at Wake Forest University School of Medicine (WFUSM) in a pathogen-free facility of the Animal Research Program under 12:12-h light/dark cycle and fed ad libitum. Both male and female homozygous mice were used, and their ages ranged from 3 to 5 months. Mice were sacrificed by cervical dislocation after isoflurane anesthesia. Ethics Statement Animal handling and procedures were approved by WFUSM Animal Care and Use Committee protocol A10-159. Cell cultures A pool of hindlimb muscles; flexor digitorum brevis (FDB) muscle only; extensor digitorum longus (EDL) muscle satellite cell culture; and primary Schwann cells were used as indicated for each experiment. The pooled hindlimb muscles were used when a large number of mesenchymal cells was needed. The FDB muscle was preferred over more traditional muscles for most experiments because it is small and flat, allowing more complete dissociation by trituration in a single step, and shortening the experiment significantly. EDL myofibers were used to isolate exclusively nestin-GFP+ satellite cells. The primary Schwann cell culture was used as a positive control for the S100b antibody. All muscle- derived cultures were grown on laminin-coated coverslips in tissue culture plates. Pooled hindlimb muscle preparation. The preparation includes all hindlimb muscles except for those in the foot. It was used for FACS on the day of muscle dissociation or at later times, followed by cell culture, as explained below. Hindlimb muscles were carefully dissected away from the surrounding connective tissue and minced. They were digested by gentle agitation in 0.2% (w/v) type-2 collagenase in Krebs solution at 37uC for 2 hours, then dissociated by trituration and resuspension in 0.25% trypsin/ 0.05% EDTA in PBS for 15 minutes at 37uC. After centrifuging at1500 rpm for 5 minutes, the supernatant was removed, and the pellet resuspended in resuspension medium consisting of Dulbecco’s modified Eagle’s medium (DMEM), containing 2 mM L- glutamine and 1% penicillin/streptomycin, supplemented with 10% (v/v) horse serum (Invitrogen, Eugene, OR) and 0.5% (v/v) chicken embryo extract (CEE, Gemini Bio-products, West Sacramento, CA). Suspended cells were passed through a 40 mm cell strainer (BD Biosciences, Mississauga, Ontario, Canada) and used for FACS and culture. For FACS experiments performed 7–14 days after muscle dissociation, cultured nestin-GFP+ cells were washed with phosphate-buffered saline (PBS) and treated with 0.25% trypsin/ 0.05% EDTA to isolate them in a suspension. When all cells were detached, the enzymatic reaction was stopped with the resuspension medium described above. We applied mechanical trituration using fire-polished glass pipettes to increase cell dissociation. Cells were centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in DMEM at 106 cells/ml. Aggregates were removed by passing them through a 40 mm cell strainer prior to sorting. Skeletal Muscle Neural Precursor Flexor digitorum brevis (FDB) preparation. FDB muscle from nestin-GFP transgenic and wild-type mice was used for most experiments in this work. Muscles were carefully dissected away from the surrounding connective tissue and digested by gentle agitation in 0.2% (w/v) Worthington’s type-2 collagenase in Krebs solution at 37uC for 2 hours. They were resuspended in resuspension medium and dissociated by gentle trituration. The growth medium used to plate cell cultures consisted of DMEM-high glucose (Invitrogen), supplemented with 2% L-glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin, and 10% (v/v) horse serum (Invitrogen) and 0.5% (v/v) CEE (Gemini Bio-products) [18]. It supported both proliferation and differentiation of myogenic cells [18]. Extensor digitorum longus (EDL) muscle satellite cell culture. EDL muscles were carefully dissected from tendon to tendon; connective tissue and fat were removed, and collagenase digested, as described above. Single myofibers were dissociated by gentle trituration with a fire-polished Pasteur pipette. Single, intact myofibers exhibiting associated GFP+ satellite cells and with no other fluorescence cells/debris attached were transferred through a series of four 60 mm Petri dishes filled with DMEM to slough loose interstitial GFP+ cells. Myofibers (50–80) were treated with trypsin/EDTA 0,125% for 15 minutes at 37uC and filtered through a 40 mm cell strainer. Filtered cells were cultured in F10 Ham medium (Invitrogen) supplemented with 20% FBS, 2 mM L- glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin (Invitrogen), and 5 ng/ml basic Fibroblast Growth Factor (Promega, Madison, WI). Primary Schwann cell culture. Primary mouse Schwann cells, isolated from early postnatal (P2-P8) C57BL/6 mouse sciatic nerves, were purchased from ScienCell Research Laboratories and cultured in Schwann cell medium (ScienCell Research Lab, Carlsbad, CA). They were plated on 35-mm dishes coated with 50 mg/ml poly-L-lysine following the company’s protocol. Immunocytochemistry, skeletal muscle histology, and cell-proliferation assay For immunostaining, culture cells were fixed with 4% paraformaldehyde (PFA) for 30 minutes, then permeabilized in 0.5% Triton X-100 (Sigma, St. Louis, MO), and blocked to saturate nonspecific antigen sites using 5% (v/v) goat serum/PBS (Jackson Immunoresearch Labs, West Grove, PA) overnight at 4uC. The next day, the cells were incubated with primary antibodies at room temperature for 4 h and visualized using appropriate species-specific secondary antibodies conjugated with Alexa Fluor 488, 568, or 647 at 1:1000 dilution (Invitrogen). They were counterstained with Hoechst 33342 reagent at 1:2000 dilution (Invitrogen) to label the DNA and mounted on slides for fluorescent microscopy with Fluorescent Mounting Medium (DakoCytomation, Carpinteria, CA). For histological analysis of nestin-GFP and Pax7 expression in the nestin-GFP transgenic mouse, we used EDL muscle, whose fibers, in contrast to FDB, exhibit a simple pennation. The complexity of FDB fiber pennation precludes sharp, uniform perpendicular cross-sections. EDL muscles from nestin-GFP transgenic mice were dissected, prefixed in 2% PFA in 0.1 M of PBS at pH 7.4 for 24 h at 4uC, and cryoprotected with sucrose, as described [19]. Muscles were permeabilized with 0.5% Triton X- 100 in PBS, blocked with 10% goat serum at 4uC overnight, exposed to Pax7 antibody (1/100 dilution) plus 1% goat serum for 3 h and then to the goat anti-mouse Alexa Fluor 568 (1/1000) secondary antibody. Hoechst 33342 (1/2000) and Fluorescent Mounting Medium were used as described above for cultured cells. PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e16816