We would like to thank Tom Cheung (The Hong Kong University of Science &#38; Technology) for advice on MuSC isolation. This work was funded by the NIAMS-IRP through NIH grants AR041126 and AR041164.

<ol>
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I., Yamamoto, N., Takeda, S., Tsuchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+progenitors+distinct+from+satellite+cells+contribute+to+ectopic+fat+cell+formation+in+skeletal+muscle.">Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle.</a> Nature Cell Biology. 12 (2), 143-152 (2010).</li><li>Wosczyna, M. N., Biswas, A. A., Cogswell, C. A., Goldhamer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+progenitors+resident+in+the+skeletal+muscle+interstitium+exhibit+robust+BMP-dependent+osteogenic+activity+and+mediate+heterotopic+ossification.">Multipotent progenitors resident in the skeletal muscle interstitium exhibit robust BMP-dependent osteogenic activity and mediate heterotopic ossification.</a> Journal of Bone and Mineral Research. 27 (5), 1004-1017 (2012).</li><li>Eisner, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+tissue-resident+PDGFR&#945;++fibro-adipogenic+progenitors+spontaneously+acquire+osteogenic+phenotype+in+an+altered+inflammatory+environment.">Murine tissue-resident PDGFR&#945;+ fibro-adipogenic progenitors spontaneously acquire osteogenic phenotype in an altered inflammatory environment.</a> Journal of Bone and Mineral Research. 35 (8), 1525-1534 (2020).</li><li>Biferali, B., Proietti, D., Mozzetta, C., Madaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro-adipogenic+progenitors+cross-talk+in+skeletal+muscle:+The+social+network.">Fibro-adipogenic progenitors cross-talk in skeletal muscle: The social network.</a> Frontiers in Physiology. 10, 1074 (2019).</li><li>Lemos, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nilotinib+reduces+muscle+fibrosis+in+chronic+muscle+injury+by+promoting+TNF-mediated+apoptosis+of+fibro/adipogenic+progenitors.">Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors.</a> Nature Medicine. 21 (7), 786-794 (2015).</li><li>Malecova, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+cellular+states+of+fibro-adipogenic+progenitors+during+myogenesis+and+muscular+dystrophy.">Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy.</a> Nature Communications. 9, 3670 (2018).</li><li>Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nature Protocols. 10 (10), 1612-1624 (2015).</li><li>Maesner, C. C., Almada, A. E., Wagers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Established+cell+surface+markers+efficiently+isolate+highly+overlapping+populations+of+skeletal+muscle+satellite+cells+by+fluorescence-activated+cell+sorting.">Established cell surface markers efficiently isolate highly overlapping populations of skeletal muscle satellite cells by fluorescence-activated cell sorting.</a> Skeletal Muscle. 8, 6-35 (2016).</li><li>Low, M., Eisner, C., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors+(FAPs):+Isolation+by+FACS+and+culture.">Fibro/adipogenic progenitors (FAPs): Isolation by FACS and culture.</a> Methods and Protocols. 1556, 179-189 (2017).</li><li>Judson, R. N., Low, M., Eisner, C., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+differentiation+of+fibro/adipogenic+progenitors+(FAPs)+from+skeletal+muscle.">Isolation, culture, and differentiation of fibro/adipogenic progenitors (FAPs) from skeletal muscle.</a> Methods in Molecular Biology. 1668, 93-103 (2017).</li><li>Hamilton, T. G., Klinghoffer, R. A., Corrin, P. 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Establishing efficient and reproducible protocols for the identification and isolation of pure adult stem cell populations is the first and most critical step toward understanding their function. Isolated FAPs and MuSCs can be used to conduct multiomics analysis in transplantation experiments as a potential treatment for muscular diseases or can be genetically modified for disease modeling in stem cell therapy. 
The protocol described here provides standardized guidelines for the identification, isolation, and culture of FAPs and MuSCs obtained from hind limb muscles of adult mice. Pure populations of FAPs and MuSCs were isolated using a FACS-based technique, and their purity was subsequently assessed by immunostaining cells with specific cell surface markers and genetic means.
The protocol consists of three main sections that highly impact the yield of FAPs and MuSCs: the mechanical and enzymatic muscle digestion, the generation of a mononucleated cell suspension through the 20 G needle, and the final isolation of single cells through FACS. 
Performing proper muscle mincing is of critical importance to release single cells. Emphasis should be placed on this step, as reaching the optimal size of the muscle pieces after mincing allows the enzymes used for digestion to work best and prevents clogging the needle used in step 4.1. On the other hand, over-mincing the muscle may result in poor cell yield and reduced cell viability, as MuSCs are rapidly released during the first digestion and may be aspirated in step 3.7.2 or 3.8.214. Additionally, the efficiency of the enzymes used for the digestion of the muscle preparation also impacts the quality of the enzymatic dissociation, as there may be variability among different enzyme lots or a decrease in enzymes' activity with time23. Therefore, it is advised to test each batch of enzymes to optimize the digestion timing and concentration. Furthermore, the concentration and amount of time required for digesting the muscle may also be affected by pathological conditions that affect muscle stiffness. These particular conditions will require individual optimization. 
The final generation of mononucleated cells happens by running the suspension through a 10 mL syringe with a 20 G needle. Specific care should be taken when performing this step to minimize the number of bubbles formed as their bursting causes additional cell damage24. 
The cell suspension is then stained with an antibody cocktail and acquired with a cell sorter. Setting the proper gates is another critical step. When performing these experiments, it is strongly recommended to run the listed single stained controls and FMO controls to properly compensate for emission spillover and account for the background signal due to spillover spread. This is especially important when dealing with bright fluorescent proteins like GFP and indirect stains like the VCAM-1-biotin/Stretpavidin-PE-Cy7 complex.
Moreover, before executing this experiment for the first time, it is good practice to perform a titration of the antibodies used to detect populations of interest. Determining the optimal concentration of the antibodies is a critical step to ensuring the brightest signal of the positive population and avoiding the increase in background staining. 
Once the sorting is completed, it is important to perform a post-sort analysis on the sorted cells to determine their purity and viability. The yield and viability of the sorted cells are highly influenced by the amount of time required to process the sample and should be taken into consideration when planning to process multiple mice. Furthermore, keeping the sorted cells on ice in 2x serum-enrich media can help cell recovery and improve their viability.
In this protocol, FAPs and MuSCs were isolated from the tibialis anterior muscles of uninjured mice as well as 7 days after Notexin injection. Following an acute injury, different FAP and MuSC sub-populations have been reported to be transiently expressed in the regenerating muscle tissue. Malecova and colleagues have reported the presence of a sub-population of VCAM-1 expressing FAPs that is absent in undamaged muscle, peaked between days 2 and 3 post-injury in acute inflammation, and persisted in murine dystrophic mice13. Similarly, Kafadar and colleagues have reported a transient increase in the expression of Sca1 on a small subset of myogenic progenitors 2 days post injury25. However, Sca1+ myogenic cells were greatly decreased 3 days after injury25. The presence of these subpopulations should be acknowledged and considered when using this protocol to isolate FAPs and MuSCs at earlier stages.
In summary, this protocol describes a method for the isolation and culture of a pure population of FAPs and MuSCs isolated from either healthy or injured adult mouse muscles. The high purity and viability of the cells make this protocol suitable for further downstream applications.

The skeletal muscle is the largest tissue in the body, accounting for ~40% of adult human weight, and is responsible for maintaining posture, generating movement, regulating basal energy metabolism, and body temperature1. Skeletal muscle is a highly dynamic tissue and possesses a remarkable ability to adapt to a variety of stimuli, such as mechanical stress, metabolic alterations, and daily environmental factors. In addition, skeletal muscle regenerates in response to acute injury, leading to complete restoration of its morphology and functions2. Skeletal muscle plasticity mainly relies upon a population of resident muscle stem cells (MuSCs), also termed satellite cells, which are located between the myofiber plasma membrane and the basal lamina2,3. Under normal conditions, MuSCs reside in the muscle niche in a quiescent state, with only a few divisions to compensate for cellular turnover and to replenish the stem cell pool4. In response to injury, MuSCs enter the cell cycle, proliferate, and either contribute to the formation of new muscle fibers or return to the niche in a self-renewal process2,3. In addition to MuSCs, homeostatic maintenance and regeneration of the skeletal muscle rely upon the support of a population of muscle resident cells named fibro-adipogenic progenitors (FAPs)5,6,7. FAPs are mesenchymal stromal cells embedded in the muscle connective tissue and capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage5,8,9,10. FAPs provide structural support for MuSCs as they are a source of extracellular matrix proteins in the muscle stem cell niche. FAPs also promote long-term maintenance of the skeletal muscle by secreting cytokines and growth factors that provide trophic support for myogenesis and muscle growth6,11. Upon acute muscle injury, FAPs rapidly proliferate to produce a transient niche that supports the structural integrity of the regenerating muscle and provides a favorable environment to sustain MuSCs proliferation and differentiation in a paracrine manner5. As regeneration proceeds, FAPs are cleared from the regenerative muscle by apoptosis, and their numbers gradually return to basal level12. However, in conditions favoring chronic muscle injury, FAPs override pro-apoptotic signaling and accumulate in the muscle niche, where they differentiate into collagen-producing fibroblasts and adipocytes, leading to ectopic fat infiltrates and fibrotic scar formation12,13.
Due to their multipotency and their regenerative abilities, FAPs and MuSCs have been identified as prospective targets in regenerative medicine for the treatment of skeletal muscle disorders. Therefore, to investigate their function and therapeutic potential, it is important to establish efficient and reproducible protocols for the isolation and culture of FAPs and MuSCs.
Fluorescence-activated cell sorting (FACS) can identify different cell populations based on morphological characteristics such as size and granularity, and permits cell-specific isolation based on the use of antibodies directed against cell surface markers. In adult mice, MuSCs express the vascular cell adhesion molecule 1 (VCAM-1, also known as CD106)14,15 and &#945;7-Integrin15, while FAPs express the platelet-derived growth factor receptor &#945; (PDGFR&#945;) and the stem cell antigen 1 (Sca1 or Ly6A/E)5,6,9,12,16,17. In the protocol described here, MuSCs were identified as CD31-/CD45-/Sca1-/VCAM-1+/&#945;7-Integrin+, while FAPs were identified as CD31-/CD45-/Sca1+/VCAM-1-/&#945;7-Integrin-. Alternatively, PDGFR&#945;EGFP mice were employed to isolate FAPs as CD31-/CD45-/PDGFR&#945;+/VCAM-1-/&#945;7-Integrin- events18,19. Furthermore, we compared the overlapping between the fluorescent signal of PDGFR&#945;-GFP+ cells to cells identified by the surface marker Sca1. Our analysis showed that all GFP-expressing cells were also positive for Sca1, indicating that either approach can be employed for the identification and isolation of FAPs. Finally, staining with specific marker antibodies confirmed the purity of each cell population.

<table><tbody><tr><td>5 mL Polypropylene Round-Bottom Tube</td><td>Falcon</td><td>352063</td><td></td></tr><tr><td>5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>20 G BD Needle 1 in. single use, sterile</td><td>BD Biosciences&amp;nbsp;</td><td>305175</td><td></td></tr><tr><td>anti-Alpha 7 Integrin PE (clone:R2F2) (RatIgG2b)</td><td>The University of British Columbia</td><td>53-0010-01</td><td></td></tr><tr><td>APC anti-mouse CD31 Antibody</td><td>BioLegend</td><td>102510</td><td></td></tr><tr><td>APC anti-mouse CD45 Antibody</td><td>BioLegend</td><td>103112</td><td></td></tr><tr><td>BD FACSMelody Cell Sorter</td><td>BD Biosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>BD Luer-Lok tip control syringe, 10-mL</td><td>BD Biosciences&amp;nbsp;</td><td>309604</td><td></td></tr><tr><td>Biotin anti-mouse CD106 Antibody</td><td>BioLegend</td><td>105703</td><td></td></tr><tr><td>C57BL/6J&amp;nbsp; mouse (Female and Male)</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>B6.129S4-Pdgfratm11(EGFP)Sor/J mouse</td><td>The Jackson Laboratory</td><td>007669</td><td></td></tr><tr><td>Corning BioCoat Collagen I 6-well Clear Flat Bottom TC-treated Multiwell Plate</td><td>Corning</td><td>356400</td><td></td></tr><tr><td>Corning BioCoat Collagen I 12-well Clear Flat Bottom TC-treated Multiwell Plate</td><td>Corning</td><td>356500</td><td></td></tr><tr><td>Corning BioCoat Collagen I 24-well Clear Flat Bottom TC-treated Multiwell Plate</td><td>Corning</td><td>356408</td><td></td></tr><tr><td>DAPI Solution (1 mg/mL)</td><td>ThermoFisher Scientific</td><td>62248</td><td></td></tr><tr><td>Disposable Aspirating Pipets, Polystyrene, Sterile</td><td>VWR</td><td>414004-265</td><td></td></tr><tr><td>Donkey anti-Goat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>ThermoFisher Scientific</td><td>A-11055</td><td></td></tr><tr><td>Falcon 40 &amp;micro;m Cell Strainer, Blue, Sterile</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Falcon 60 mm TC-treated Cell Culture Dish, Sterile</td><td>Corning</td><td>353002</td><td></td></tr><tr><td>Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 15-mL</td><td>VWR</td><td>352196</td><td></td></tr><tr><td>Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 50-mL</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>Falcon Round-Bottom Tubes, Polypropylene, Corning</td><td>VWR</td><td>60819-728</td><td></td></tr><tr><td>Falcon Round-Bottom Tubes, Polystyrene, with 35um Cell Strainer Cap Corning</td><td>VWR</td><td>21008-948</td><td></td></tr><tr><td>Fibroblast Growth Factor, Basic, Human, Recombinant (rhFGF, Basic)</td><td>Promega</td><td>G5071</td><td></td></tr><tr><td>FlowJo 10.8.1</td><td></td><td></td><td></td></tr><tr><td>Gibco Collagenase, Type II, powder</td><td>ThermoFisher Scientific</td><td>17101015</td><td></td></tr><tr><td>Gibco Dispase, powder</td><td>ThermoFisher Scientific</td><td>17105041</td><td></td></tr><tr><td>Gibco DMEM, high glucose, HEPES</td><td>ThermoFisher Scientific</td><td>12430054</td><td></td></tr><tr><td>Gibco Fetal Bovine Serum, certified, United States</td><td>ThermoFisher Scientific</td><td>16000044</td><td></td></tr><tr><td>Gibco Ham&#039;s F-10 Nutrient Mix</td><td>ThermoFisher Scientific</td><td>11550043</td><td></td></tr><tr><td>Gibco Horse Serum, New Zealand origin</td><td>ThermoFisher Scientific</td><td>16050122</td><td></td></tr><tr><td>Gibco PBS, pH 7.4</td><td>ThermoFisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Gibco PBS (10x), pH 7.4</td><td>ThermoFisher Scientific</td><td>70011044</td><td></td></tr><tr><td>Gibco Penicillin-Streptomycin-Glutamine (100x)</td><td>ThermoFisher Scientific</td><td>10378016</td><td></td></tr><tr><td>Goat anti-Mouse IgG1 cross-absorbed secondary antibody, Alexa Fluor 555</td><td>ThermoFisher Scientific</td><td>A-21127</td><td></td></tr><tr><td>Hardened Fine Scissors</td><td>Fine Science Tools Inc</td><td>14090-09</td><td></td></tr><tr><td>Invitrogen 7-AAD (7-Aminoactinomycin D)</td><td>ThermoFisher Scientific</td><td>A1310</td><td></td></tr><tr><td>Mouse PDGF&amp;nbsp;R alpha Antibody</td><td>R&amp;amp;D Systems</td><td>AF1062</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Fisher Scientific</td><td>NC9624464</td><td></td></tr><tr><td>Normal Goat Serum</td><td>ThermoFisher Scientific</td><td>31872</td><td></td></tr><tr><td>Pacific Blue anti-mouse Ly-6A/E (Sca 1) Antibody</td><td>BioLegend</td><td>108120</td><td></td></tr><tr><td>Paraformaldehyde, 16%</td><td>Fisher Scientific</td><td>NCC0528893</td><td></td></tr><tr><td>Pax7 mono-clonal mouse antibody (IgG1) (supernatant)</td><td>Developmental Study Hybridoma Bank</td><td>N/A</td><td></td></tr><tr><td>PE/Cyanine7 Streptavidin</td><td>BioLegend</td><td>405206</td><td></td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools Inc</td><td>91500-09</td><td></td></tr><tr><td>Student Dumont #5 Forceps</td><td>Fine Science Tools Inc</td><td>91150-20</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr></tbody></table>

facs-isolation and culture of fibro-adipogenic progenitors and muscle stem cells from unperturbed and injured mouse skeletal muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

This protocol allows the isolation of approximately one million FAPs and up to 350,000 MuSCs from uninjured hind limbs of wild-type adult mice (3-6 months), corresponding to a yield of 8% for FAPs and 3% for MuSCs of total events. When sorting cells from damaged TA 7 days post-injury, two to three TA muscles are pooled to obtain up to 300,000 FAPs and 120,000 MuSCs, which correspond to a yield of 11% and 4%, respectively. Post-sort purity values are usually above 95% for FAPs and MuSCs.
The gating strategy adopted to isolate FAPs and MuSCs is illustrated in Figure 2. First, cell populations of interest are identified by creating a forward scatter (FSC) versus side scatter (SSC) density plot, which allows for some degree of cellular identification based on cell morphological properties. This gating strategy is also used to exclude small cellular debris, which is usually located at the bottom left corner of the FSC vs SSC plot. Cells are further gated to exclude doublets based on FSC and SSC height and width signals, and the resulting singlet cells are then stained with 7-AAD to evaluate their viability. Living cells are further assessed for Sca1 and CD31/CD45 expression to exclude Lineage positive cells from further analysis. Lineage negative Sca1 negative singlets are then stained for VCAM-1 and &#945;7-Integrin to distinguish FAPs and MuSCs. FAPs are identified as CD31-/CD45-/Sca1+/VCAM-1-/&#945;7-Integrin- cells and MuSCs are identified as CD31-/CD45-/Sca1-/VCAM-1+/&#945;7-Integrin+ cells. Alternatively, living cells are also gated for CD31/CD45 and GFP-PDGFR&#945; to distinguish FAPs. FAPs are identified as CD31-/CD45-/PDGFR&#945;+/VCAM-1-/&#945;7-Integrin- cells.
This protocol presents two gating strategies to isolate the FAP population: either by using an endogenous PDGFR&#945;-EGFP reporter or by performing cell staining with a Sca1 antibody. The PDGFR&#945;-EGFP knock-in reporter mice (B6.129S4-Pdgfratm11(EGFP)Sor/J)18 express the H2B-eGFP fusion gene from the endogenous PDGFR&#945; locus, allowing efficient and specific labeling of the PDGFR&#945; lineage (Figure 3A). This mouse line was previously used to isolate Pdgfr&#945;+ FAPs and is indeed very helpful for the isolation of FAPs, as the GFP reporter provides a highly visible and specific signal19. Alternatively, this protocol describes a Sca1-based isolation method for the identification of FAPs. Sca1 (Ly-6A/E) is commonly used to identify and isolate FAPs in muscle preparation13,16,17. Sca1 is an 18-kDa glycosylphosphatidylinositol (GPI)-linked protein member of the Ly-6 family20. However, besides being expressed on the cell surface of mesenchymal progenitor muscle cells, Sca1 expression is also observed in hematopoietic stem cells (HSCs) as well as mature leukocytes and T cells. In this protocol, we confirmed the suitability of Sca1 as FAPs&#39; marker by performing FACS-based lineage tracing studies. Specifically, PDGFR&#945;-EGFP mice were employed to isolate Sca1+/GFP-expressing FAPs among populations of mononucleated cells. We found that all GFP-expressing cells were also positive for Sca1 and negative for CD31, CD45, VCAM-1, and &#945;7-Integrin (Figure 4). This analysis confirmed the use of Sca1 antibody as a marker for FAPs in both quiescent and activated FAPs and provides means for the indistinct use of either strategy to isolate FAPs.
In this protocol, FAPs and MuSCs were also isolated from adult mice injured with intramuscular injections of Notexin, 7 days post-injury (Figure 5). Following injury, MuSCs and FAPs activate and proliferate in vivo, reaching the peak of proliferation between day 3 and 42,3. These changes in proliferation are reflected by an increase in the percentage of Sca1/PDFGR&#945; as well as VCAM-1/&#945;7-Integrin positive cells. Figure 6 represents the quantification of FAPs and MuSCs in uninjured and 7 days post-injury TAs; although the peak in proliferation is observed between days 2 and 3 after injury, a low rate of proliferating cells is still appreciable 7 days after injury. As MuSCs are activated, there is a marked increase in their cellular size21,22. Therefore, while isolating these cells by FACS, it is of critical importance to adjust FSC-A and SSC-A parameters and expand the gate to include those cells in the center (Figure 5A).
The purity of the isolated cell populations was confirmed by immunostaining of cocultured GFP+ FAPs and MuSCs with Pax7 antibodies. Freshly isolated GFP+ FAPs and MuSCs were cocultured for 48 h at a ratio of 1:1 (Figure 3B). GFP+ FAPs did not express Pax7 marker, while MuSCs reacted with this antibody. Thus, Lin-/Sca-1+/VCAM-1-/&#945;7-Integrin- and Lin-/Sca-1-/VCAM-1+/&#945;7-Integrin+ gating strategy is effective in isolating pure populations of FAPs and MuSCs, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63983/63983fig01.jpg" /> 
Figure 1: Graphical overview of FAP and MuSC isolation. Graphical overview showing FAP and MuSC isolation from hind limb muscles. The protocol also applies to cells isolated from TA muscles. First, muscles are collected and mechanically minced before undergoing enzymatic digestion. The muscle mixture is then processed through a 20 G needle and filtered to obtain a suspension of mononucleated cells. Cells are then incubated with a cocktail of fluorophore-conjugated antibodies and ultimately run through a cell sorter to isolate a pure population of FAPs and MuSCs. Pure cell populations are then processed for downstream application. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63983/63983fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63983/63983fig02.jpg" /> 
Figure 2: Representative FACS profile of quiescent FAPs and MuSCs. (A-H) Gating strategy for the isolation of FAPs and MuSCs. (A) First, samples are gated to exclude cellular debris and (B,C) doublets based on SSC and FSC properties. (D) The resulting single cells are then stained with 7-AAD to evaluate their viability. (E) 7-AAD negative single cells are then assessed for APC-CD31/CD45 and Pacific Blue-Sca1 to exclude Lineage positive cells from further analysis. (F,G) Lineage negative singlets are then stained for PE-Cy7-VCAM-1 and PE-&#945;7-Integrin. (F) FAPs are identified as CD31-/CD45-/Sca1+/VCAM-1-/&#945;7-Integrin- cells and (G) MuSCs are identified as CD31-/CD45-/Sca1-/VCAM-1+/&#945;7-Integrin+ cells. (H) FAPs isolation in PDGFR&#945;-EGFP mice using a GFP reporter. (I-M) FACS profile for Fluorescence Minus One (FMO) controls to demonstrate proper compensation and gating. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63983/63983fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63983/63983fig03.jpg" /> 
Figure 3: Validation of FAP and MuSC cell culture. (A) Freshly isolated FAPs expressing GFP were plated and co-stained with a PDGFR&#945; antibody. Nuclei were stained with DAPI. Merged images of GFP (green), PDGFR&#945; (red), and DAPI (blue) staining are displayed. Scale bars, 25 &#956;m. (B) Freshly isolated FAPs (green) and MuSCs were cocultured for 48h and stained with Pax7 (red). Nuclei were stained with DAPI. GFP-expressing FAPs do not express Pax7, while MuSCs exclusively express Pax7 and are not positive for GFP, confirming the purity of both sorted populations. Scale bars, 75 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63983/63983fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63983/63983fig04.jpg" /> 
Figure 4: PDGFR&#945;-EGFP mice and Sca1 expression specifically label FAPs in adult mice. FAPs are isolated as GFP+/Sca1+ cells. 7-AAD negative single cells are assessed for APC-CD31/CD45 and GFP-PDGFR&#945; to exclude Lineage positive cells and distinguish FAPs. Lineage negative/Sca1 positive cells are stained for PE-Cy7-VCAM-1 and PE-&#945;7-Integrin to exclude MuSCs. FAPs are identified as CD31-/CD45-/PDGFR&#945;+/Sca1+/VCAM-1-/&#945;7-Integrin- cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63983/63983fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63983/63983fig05.jpg" /> 
Figure 5: Representative FACS profile of activated FAPs and MuSCs. (A-H) Gating strategy for the isolation of activated FAPs and MuSCs. (A) Samples are gated to exclude cellular debris by creating FSC-A vs SSC-A density plot. Note the enlarged gate to accommodate the population of interest. (B,C) Samples are further gated to eliminate doublets based on SSC and FSC properties. (D) The resulting single cells are then stained with 7-AAD to evaluate their viability. (E) 7-AAD negative single cells are then assessed for APC-CD31/CD45 and Pacific Blue-Sca1 to exclude Lineage positive cells from further analysis. (F,G) Lineage negative singlets are then stained for PE-Cy7-VCAM-1 and PE-&#945;7-integrin. (F) FAPs are identified as CD31-/CD45-/Sca1+/VCAM-1-/&#945;7-integrin- cells and (G) MuSCs are identified as CD31-/CD45-/Sca1-/VCAM-1+/&#945;7-integrin+ cells. (H) FAPs isolation in PDGFR&#945;-EGFP mice using a GFP reporter. (I-M) FACS profile for Fluorescence Minus One (FMO) controls to demonstrate proper compensation and gating. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63983/63983fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63983/63983fig06.jpg" /> 
Figure 6: Quantification of FAPs and MuSCs in unperturbed and injured TAs. Quantification of FAPs and MuSCs in uninjured and 7 days post-injury TA muscles. Cell counting was performed by dividing the number of cells sorted per mgs of muscle collected. ** P &#60; 0.01 between uninjured vs injured. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63983/63983fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2"></td>
			<td>7-AAD 
			(&#181;g/mL)</td>
			<td>CD31/CD45-APC 
			(&#181;g/mL)&#160;</td>
			<td>Sca1-Pacific Blue 
			(&#181;g/mL)</td>
			<td>&#945; 7-Integrin-PE 
			(&#181;g/mL)</td>
			<td>VCAM-1-Biotin 
			(&#181;g/mL)</td>
			<td>PE-Cy7 Streptavidin 
			(&#181;g/mL)</td>
		</tr>
		<tr>
			<td colspan="2">Unstained Control</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Single Stained Controls</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">7-AAD (Viability Control)</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">CD31/CD45</td>
			<td></td>
			<td>2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Sca1</td>
			<td></td>
			<td></td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">&#945;7-Integrin</td>
			<td></td>
			<td></td>
			<td></td>
			<td>1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">VCAM-1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>5</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="2">FMO Controls</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">FMO 7 AAD</td>
			<td></td>
			<td>2</td>
			<td>5</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="2">FMO CD31/CD45</td>
			<td>1</td>
			<td></td>
			<td>5</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="2">FMO Sca1</td>
			<td>1</td>
			<td>2</td>
			<td></td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="2">FMO &#945;7-Integrin</td>
			<td>1</td>
			<td>2</td>
			<td>5</td>
			<td></td>
			<td>5</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="2">FMO VCAM-1</td>
			<td>1</td>
			<td>2</td>
			<td>5</td>
			<td>1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Experimental sample</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Full stain</td>
			<td>1</td>
			<td>2</td>
			<td>5</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Antibody staining matrix. List of antibody concentrations used for staining the experimental and control samples. If isolating FAPs by GFP, Sca1 staining can be omitted. Instead, run GFP single stained control and GFP FMO.

Watch this Scientific Journal Video about FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle at JoVE.com

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

facs-isolation-culture-fibro-adipogenic-progenitors-muscle-stem-cells

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.0K Views.  National Institutes of Health (NIH). Dit protocol beschrijft een methode voor de gelijktijdige isolatie van fibro adipogene, voorlopercellen en spierstamcellen met behulp van fluorescentieactivatoren van sorteren. Zuivere populaties van deze cellen zijn een voorwaarde voor het begrijpen van hun biologische rol in fysiologische en pathologische omstandigheden. Met behulp van deze techniek werden Forbes en spierstamcellen geïdentificeerd en geïsoleerd uit oksel of gewonde spieren.De hoge gevulde straffeloosheid van Forbes...

Video: FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Biology

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mouse Heart and Skeletal Muscle Digestion for Fibro-Adipogenic Progenitor Culture

Fibro-Adipogenic Progenitor Isolation, Expansion, and Differentiation from the Spiny Mouse Model

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are tissue-resident cells that are able to differentiate into adipocytes, fibroblasts, and chondrocytes. Fibro-adipogenic progenitors are fundamental for orchestrating tissue regeneration as they are responsible for extracellular matrix remodeling after injury. This study focuses on investigating the specific role of fibro-adipogenic progenitors in spiny mouse cardiac repair and skeletal muscle regeneration. To this end, a protocol has been optimized for the purification of spiny mouse fibro-adipogenic progenitors by flow cytometry from enzymatically dissociated skeletal and cardiac muscle. The population obtained from this protocol is capable of expanding in vitro, and can be differentiated to myofibroblasts and adipocytes. This protocol offers a valuable tool for researchers to examine the distinctive properties of spiny mouse, and to compare them to the Mus musculus. This will provide insights that could advance the understanding of regenerative mechanisms in this intriguing model.

Author Spotlight: Streamlining Cell Analysis with an Innovative Protocol for Studying Spiny Mouse Models

This protocol outlines the isolation of skeletal and cardiac muscle fibro-adipogenic progenitors (FAPs) from spiny mouse (Acomys) via enzymatic dissociation and fluorescence-activated cell sorting. The FAPs obtained from this protocol can be effectively expanded and differentiated to myofibroblasts and adipocytes.

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Medicine

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation assay.

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2&#180;-deoxyuridine incorporation assay.

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

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