Nestin-GFP transgenic mice were maintained homozygous for the transgene on the C57BL/6 genetic background (Mignone et al., 2004
). C57BL/6 wild-type mice were used as controls. NG2-DsRed transgenic mice expressing DsRed-T1 under the control of the NG2 promoter (Zhu et al., 2008
) were purchased from the Jackson Laboratory. Nestin-GFP mice were crossbred with NG2-DsRed mice to generate Nestin-GFP/NG2-DsRed double-transgenic mice. All mouse colonies were housed at Wake Forest School of Medicine (WFSM) 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. Animal handling and procedures were approved by the WFSM Animal Care and Use Committee.
Flexor digitorum brevis (FDB) muscle culture preparation
FDB muscle from Nestin-GFP transgenic, NG2-DsRed transgenic, Nestin-GFP/NG2-DsRed transgenic, and C57BL/6 wild-type mice were used for most experiments in this work. 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, shortening the experiment significantly (Zhang et al., 2011
). Methods for FDB culture preparation have been described (Birbrair et al., 2011
). Briefly, muscles were carefully dissected away from the surrounding connective tissue and minced, then digested by gentle agitation in 0.2% (w/v) Worthington’s type-2 collagenase in Krebs solution at 37°C for 2 hours. They were resuspended in growth medium and dissociated by gentle trituration. The growth medium used to plate cell cultures consisted of DMEM-high glucose (Invitrogen, Carlsbad, CA, USA), supplemented with 2% L-glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin, 10% (v/v) horse serum (Invitrogen) and 0.5% (v/v) CEE (Gemini Bio-products, West Sacramento, CA, USA). It supported both proliferation and differentiation of myogenic cells (Zammit et al., 2004
Cultured cells were fixed with 4% PFA for 30 minutes, then permeabilized in 0.5% Triton X-100 (Sigma, St. Louis, MO, USA), and blocked to saturate nonspecific antigen sites using 5% (v/v) goat serum/PBS (Jackson Immunoresearch Labs, West Grove, PA, USA) overnight at 4°C. 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, 647 or 680 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, USA).
shows antibodies, their dilution, and source.
Antibodies, concentration, and source
Skeletal muscle processing
To detect DsRed and GFP fluorescence, nondissociated extensor digitorum longus (EDL) muscles from 3-month-old Nestin-GFP/NG2-DsRed mice were dissected, fixed in 4% paraformaldehyde overnight, immersed in 10%, 20%, and 30% sucrose solutions for 60, 45, and 30 minutes, respectively, embedded in OCT, and rapidly frozen in liquid nitrogen to prepare 10-μm thick cryosections. Muscle sections were fixed with 4% PFA for 30 minutes, then permeabilized in 0.5% Triton X-100 (Sigma), and blocked to saturate nonspecific antigen sites using 5% (v/v) goat serum/PBS (Jackson Immunoresearch Labs) overnight at 4°C. The next day, the sections 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, 647 or 680 at 1:1000 dilution (Invitrogen). Muscle sections were counterstained with Hoechst 33342, mounted on slides using Fluorescent Mounting Medium (DakoCytomation), and examined with fluorescence microscopy.
Microscopy, Cell Imaging, and Counting
An inverted motorized fluorescent microscope (Olympus, IX81, Tokyo, Japan) with an Orca-R2 Hamamatsu CCD camera (Hamamatsu, Japan) was used for image acquisition. Camera drive and acquisition were controlled by a MetaMorph Imaging System (Olympus, Center Valley, PA, USA). Ten arbitrary microscopic fields were counted in each immunostained plate, and values pooled from parallel duplicates per time point and individual experiment.
Fluorescence-activated cell sorting (FACS)
FACS was carried out on a BD FACS (Aria Sorter, San Jose, CA, USA) at 4°C and a pressure of 20 psi, using a laser at the 488-nm line, a 530/30 band pass filter, a 100-μm sorting tip, and a 34.2 kHz drive frequency, sterilized with 10% bleach. This instrument allowed us to characterize cells by size as well as fluorescence. Low flow rate improved sorting purity. Data acquisition and analyses were performed using BD FACS Diva 5.0.3 software, gated for a high level of GFP, DsRed, PerCP/Cy5.5 and/or APC expression. For instance, the clear separation of GFP+ from GFP- cells (Birbrair et al., 2011
), and DsRed+ from DsRed- cells explains the ease of sorting. Sorted cells were re-analyzed to confirm their fluorescence profile.
Isolation of NGFR+/Tuj1+ cells from skeletal muscle cultures of nontransgenic mice by FACS
FDB cultures from young-adult (3-5-month) C57BL/6 wild-type mice were prepared as described above. After 7 days, the cells were scraped from the dishes, dissociated by trituration and resuspended in 5 mM EDTA in PBS for 15 min at 4°C. After centrifuging at 1500 rpm for 5 min, the supernatant was removed, and the pellet resuspended in growth medium. Aggregates were removed by passing them through a 40-μm cell strainer prior to sorting. After counting, the cells were centrifuged at 1500 rpm for 5 min and resuspended in 100-μl 1% FBS in PBS /106
cells. An aliquot was used as an unlabeled control, while the remaining cells were labeled with 5 μg/mL of rabbit anti-NGFR antibody (Advanced Targeting Systems) for 1 hour. This antibody has been used to isolate NGFR+ cells from the brain (Schnitzler et al., 2008
). After one washing, the cells were labeled with APC anti-rabbit antibody (Invitrogen) for 45 minutes and, after a second washing, resuspended in 1% FBS in PBS and analyzed for forward scatter and NGFR-APC to sort out NGFR+ cells. After sorting, 2-3×104
were plated on laminin (Invitrogen) precoated dishes (Fisher Scientific, Pittsburgh, PA). Purity, neural morphology, and Tuj1 expression were analyzed after 4 days in culture.
Cell isolation from Nestin-GFP/NG2-DsRed mice skeletal muscle by FACS
A pool of hindlimb muscles, excluding those from the foot, was used for experiments that required a large number of cells as indicated. Cells were sorted immediately after skeletal muscle dissociation (time 0). Hindlimb muscles from young-adult (3-5 month) Nestin-GFP/NG2-DsRed transgenic mice were prepared as described (Birbrair et al., 2011
). Briefly, muscles were carefully dissected away from the surrounding connective tissue and minced, then digested by gentle agitation in 0.2% (w/v) type-2 collagenase in Krebs solution at 37°C for 2 hours, and dissociated by trituration and resuspension in 0.25% trypsin/0.05% EDTA in PBS for 15 minutes at 37°C. After centrifuging at 1500 rpm for 5 minutes, the supernatant was removed, and the pellet resuspended in growth medium. Aggregates were removed by passing them through a 40-μm cell strainer prior to sorting. Cells were centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, and the pellet resuspended in 1% FBS in PBS and analyzed for GFP and DsRed fluorescence to sort the different cell populations based on these two markers. Isolated Nestin-GFP+/NG2-DsRed-, Nestin-GFP-/NG2-DsRed+, Nestin-GFP+/NG2-DsRed+, and Nestin-GFP-/NG2-DsRed- cells were used for single cell RT-PCR or cultured either separately or together with FDB culture from wild-type mice. Neural morphology, and Nestin-GFP, Tuj1, and α-SMA expression were analyzed after 8 days in culture.
Single cell RT-PCR
After sorting, Nestin-GFP+/NG2-DsRed-, Nestin-GFP-/NG2-DsRed+, Nestin-GFP+/NG2-DsRed+, or Nestin-GFP-/NG2-DsRed- cells were transferred to a separate tube containing 1 ml of growth medium, homogenized and counted. The cells were diluted to obtain a concentration of 1 cell / 10 μl. The resulting cell suspension was dispensed into laminin pre-coated wells of a 24-well plate, putting only 10 μl in each well containing 200 μl of growth medium. Each well was expected to yield none or one cell per well, based on our experience with this technique previously. Cells were identified visually using an inverted motorized fluorescent microscope (Olympus). For cell harvest, fine tip transfer pipets (Fisher Scientific) coupled to a standard patch-clamp pipettes pulled from borosilicate glass (Boralex, WPI, Sarasota, FL, USA) using a Flaming Brown micropipette puller (P97, Sutter Instrument Co., Novato, CA, USA) were used. A pipette was moved into the bath solution under positive pressure. Under visual control, the tip of the pipette was gently attached to the selected cell and suction was applied, until the cell entered the tip of the pipette. The pipette was removed quickly from the bath. Under a dissecting microscope, the content of the pipette was ejected into a PCR tube, centrifuged at 1500 rpm for 5 minutes and all the solution removed. The cell was resuspended in the RNA protecting solution: (4 μl of H2O with 15 U RNAse-inhibitor (Promega, Fitchburg, WI, USA), 25 mM DTT). Then, we added 2 μl of nucleotide/detergent solution (10 mM Tris–HCl, pH 8.0, 25 μM random hexamers (Promega), 2.5 mM of each dNTP (Promega), 0.2% Nonidet P40 (Roche, Indianapolis, IN, USA), denatured at 70 °C for 5 minutes and placed it on ice for 5 minutes. After this, 2 μl of 5X First-strand buffer (Invitrogen), 0.5 μl of 200 mM DTT, 20 U RNAse-inhibitor (Promega) and 100 U superscript III reverse transcriptase (Invitrogen) were added. The RT reaction (final volume: 10 μl) was incubated at 37 °C for 1 h and stopped at 95 °C for 5 minutes. As negative controls, the RT reactions were performed in the absence of either cell (only growth medium tested) or reverse transcriptase.
For cDNA purification, the QIAEX II gel extraction Kit (Qiagen, Valencia, CA, USA) was used. The complete RT reaction (10 μl) was mixed with 80 μl QX1 binding buffer and 1.5 μl DNA-binding matrix per RT reaction at 25 °C for 15 minutes mixing every 2 minutes to keep QIAEX in suspension. The binding matrix was pelleted (13,000 rpm, 2 minutes) and washed twice in 90 μl ice-cold, ethanol-based PE-buffer (Qiagen). The DNA-binding matrix was dried at 37 °C for 10 minutes to remove the ethanol completely. Finally, the cDNA was eluted with an appropriate volume (>5 μl) of 1 mM Tris-HCl, pH 8.5 at 50 °C for 3 minutes. The binding matrix was pelleted (13,000 rpm, 2 minutes) and the supernatant containing cDNA material was stored at - 80 °C until use for PCR.
Polymerase Chain Reaction (PCR)
The cDNA was amplified by PCR using the primers included in . PCR Master Mix was purchased from Promega. Each PCR reaction contained 1× Promega PCR Master Mix, 1 μM of each primer, and the cDNA of the cell used in each case (Nestin-GFP+/NG2-DsRed-, Nestin-GFP-/NG2-DsRed+, Nestin-GFP+/NG2-DsRed+, or Nestin-GFP-/NG2-DsRed- cell). The volume of each reaction was brought up to 50 μl with water. DNA amplification was carried out as follows: denaturation at 94°C for 2 minutes, followed by 35 cycles of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes. After 35 cycles, the reactions were incubated at 72°C for 7 minutes to increase the yield of amplification. PCR products were verified with DNA 2% agarose gel electrophoresis.
Genes, GenBank Accession numbers, Coding regions, and Primers
Cell isolation using CD146-PerCP-Cy5.5 antibody from Nestin-GFP mouse skeletal muscle by FACS
Hindlimb muscle cells were isolated from young-adult (3-5-month) C57BL/6 wild-type mice as described above. After counting, cells were centrifuged at 1500 rpm for 5 min and resuspended in 100-μl 1% FBS in PBS /106 cells. An aliquot was collected for use as unlabeled control, while the remaining cells were labeled with PerCP/Cy5.5 anti-mouse CD146 antibody for 1 hour. After washing, the cells were resuspended in 1% FBS in PBS and sorted using GFP and PerCP/Cy5.5 fluorescence. Isolated Nestin-GFP+/CD146-PerCP-Cy5.5-, Nestin-GFP-/CD146-PerCP-Cy5.5+, Nestin-GFP+/CD146-PerCP-Cy5.5+, and Nestin-GFP-/CD146-PerCP-Cy5.5- cells were cultured either separately or with medium from 7-day-old FDB culture. Neural morphology and Tuj1 expression were analyzed after 8 days in culture.
Isolation of CD146+/CD31- cells from skeletal muscle by FACS
Cells isolation from hindlimb muscles from young-adult (3-5-month) C57BL/6 wild-type mice was done as described above (Birbrair et al., 2011
). To exclude endothelial cells (CD31+), magnetic separation was performed using a MACS system (Miltenyi Biotec Inc., Auburn, CA). Briefly, dissociated cells were resuspended in MACS buffer (Miltenyi) (90-μl/107
cells) and labeled with endothelial cell marker anti-CD31 beads (Miltenyi) by adding 10-μl beads/107
cells and incubating for 15 min at 4°C. After washing, the cells were resuspended in 500-μl buffer/108
cells. Magnetic separation of the cells was performed on a MACS column (Miltenyi), where CD31+ endothelial cells were retained and eluted only upon removal from the magnetic field, according to the manufacturer’s instructions. CD31- cells, obtained from MACS sorting, were centrifuged at 1500 rpm for 5 min. The supernatant was removed, and the pellet resuspended in 1% FBS in PBS. An aliquot of these cells was collected for use as unlabeled control, while the remaining cells were labeled with CD146-FITC-conjugated antibody (Miltenyi) for 1 hour. After washing, the cells were resuspended in 1% FBS in PBS and analyzed for forward scatter and CD146-FITC to sort the CD146+ and CD146- cells that were cultured either separately, together, or with medium from 7 day-old FDB culture. Neural morphology and Tuj1 expression were analyzed after 8 days in culture.
Results are expressed as the mean ± s.e.m. Statistical significance was assessed using analysis of variance (ANOVA) followed by t-test using Prism GraphPad. P < 0.05 was considered significant.