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 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.
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 α7-Integrin15, while FAPs express the platelet-derived growth factor receptor α (PDGFRα) 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+/α7-Integrin+, while FAPs were identified as CD31-/CD45-/Sca1+/VCAM-1-/α7-Integrin-. Alternatively, PDGFRαEGFP mice were employed to isolate FAPs as CD31-/CD45-/PDGFRα+/VCAM-1-/α7-Integrin- events18,19. Furthermore, we compared the overlapping between the fluorescent signal of PDGFRα-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.
All animal experiments performed were conducted in compliance with institutional guidelines approved by the Animal Care and Use Committee (ACUC) of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS). Investigators performing this protocol must adhere to their local animal ethics guidelines.
NOTE: This protocol describes in detail how to isolate FAPs and MuSCs from hind limb and injured tibialis anterior (TA) muscles of adult male and female mice (3-6 months) and provides guidelines for coculturing FAPs and MuSCs. An overview of the experimental procedure is shown in Figure 1. All steps of this protocol should be performed in sterile conditions and at room temperature (RT) unless otherwise specified.
1. Reagent setup
2. Hind limb muscle harvesting
3. Mechanical and enzymatic muscle digestion
4. Generation of mononucleated cells
NOTE: If working with TA muscles collected in a 15 mL conical tube, transfer the suspension into a 50 mL conical tube before proceeding with step 4.1.
5. Antibody staining for flow cytometry
NOTE: For each experiment, set up the following controls: i) unstained control, ii) viability control to select for the live cell population, iii) single stained compensation controls to correct for fluorochrome emission spillover, and iv) fluorescence minus one (FMO) controls to set gating boundaries by accounting for spillover spread. Refer to Table 1 for a full list of staining controls.
6. Fluorescence-activated cell sorting (FACS)
NOTE: This protocol employs a compact benchtop research flow cytometer equipped with a 100 μm nozzle and featuring a three-laser configuration (488 nm, 640 nm, 405 nm) with the capability to analyze up to nine different fluorochromes (11 parameters including the forward and side scatter). The fluorochromes used in this protocol and their associated detector bandpass filters are as follows: PE 586/42; PE-Cy7 783/56; APC 660/10; Pacific Blue 448/45; 7-Aminoactinomycin D (7-AAD) 700/54, GFP 527/32. Cells are sorted at 4 °C and remain on ice following the sort. Before operating this instrument, ensure that the user is properly trained by a technical applications specialist.
7. Culture of FAPs and MuSCs
NOTE: Sorted cells should be cultured immediately after sorting, in an appropriate medium on collagen I coated plates.
8. Immunofluorescence analysis of cultured FAPs and MuSCs
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 α7-Integrin to distinguish FAPs and MuSCs. FAPs are identified as CD31-/CD45-/Sca1+/VCAM-1-/α7-Integrin- cells and MuSCs are identified as CD31-/CD45-/Sca1-/VCAM-1+/α7-Integrin+ cells. Alternatively, living cells are also gated for CD31/CD45 and GFP-PDGFRα to distinguish FAPs. FAPs are identified as CD31-/CD45-/PDGFRα+/VCAM-1-/α7-Integrin- cells.
This protocol presents two gating strategies to isolate the FAP population: either by using an endogenous PDGFRα-EGFP reporter or by performing cell staining with a Sca1 antibody. The PDGFRα-EGFP knock-in reporter mice (B6.129S4-Pdgfratm11(EGFP)Sor/J)18 express the H2B-eGFP fusion gene from the endogenous PDGFRα locus, allowing efficient and specific labeling of the PDGFRα lineage (Figure 3A). This mouse line was previously used to isolate Pdgfrα+ 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' marker by performing FACS-based lineage tracing studies. Specifically, PDGFRα-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 α7-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/PDFGRα as well as VCAM-1/α7-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-/α7-Integrin- and Lin-/Sca-1-/VCAM-1+/α7-Integrin+ gating strategy is effective in isolating pure populations of FAPs and MuSCs, respectively.
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. Please click here to view a larger version of this figure.
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-α7-Integrin. (F) FAPs are identified as CD31-/CD45-/Sca1+/VCAM-1-/α7-Integrin- cells and (G) MuSCs are identified as CD31-/CD45-/Sca1-/VCAM-1+/α7-Integrin+ cells. (H) FAPs isolation in PDGFRα-EGFP mice using a GFP reporter. (I–M) FACS profile for Fluorescence Minus One (FMO) controls to demonstrate proper compensation and gating. Please click here to view a larger version of this figure.
Figure 3: Validation of FAP and MuSC cell culture. (A) Freshly isolated FAPs expressing GFP were plated and co-stained with a PDGFRα antibody. Nuclei were stained with DAPI. Merged images of GFP (green), PDGFRα (red), and DAPI (blue) staining are displayed. Scale bars, 25 μm. (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 μm. Please click here to view a larger version of this figure.
Figure 4: PDGFRα-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-PDGFRα to exclude Lineage positive cells and distinguish FAPs. Lineage negative/Sca1 positive cells are stained for PE-Cy7-VCAM-1 and PE-α7-Integrin to exclude MuSCs. FAPs are identified as CD31-/CD45-/PDGFRα+/Sca1+/VCAM-1-/α7-Integrin- cells. Please click here to view a larger version of this figure.
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-α7-integrin. (F) FAPs are identified as CD31-/CD45-/Sca1+/VCAM-1-/α7-integrin- cells and (G) MuSCs are identified as CD31-/CD45-/Sca1-/VCAM-1+/α7-integrin+ cells. (H) FAPs isolation in PDGFRα-EGFP mice using a GFP reporter. (I–M) FACS profile for Fluorescence Minus One (FMO) controls to demonstrate proper compensation and gating. Please click here to view a larger version of this figure.
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 < 0.01 between uninjured vs injured. Please click here to view a larger version of this figure.
7-AAD (µg/mL) |
CD31/CD45-APC (µg/mL) |
Sca1-Pacific Blue (µg/mL) |
α 7-Integrin-PE (µg/mL) |
VCAM-1-Biotin (µg/mL) |
PE-Cy7 Streptavidin (µg/mL) |
||
Unstained Control | |||||||
Single Stained Controls | |||||||
7-AAD (Viability Control) | 1 | ||||||
CD31/CD45 | 2 | ||||||
Sca1 | 5 | ||||||
α7-Integrin | 1 | ||||||
VCAM-1 | 5 | 2 | |||||
FMO Controls | |||||||
FMO 7 AAD | 2 | 5 | 1 | 5 | 2 | ||
FMO CD31/CD45 | 1 | 5 | 1 | 5 | 2 | ||
FMO Sca1 | 1 | 2 | 1 | 5 | 2 | ||
FMO α7-Integrin | 1 | 2 | 5 | 5 | 2 | ||
FMO VCAM-1 | 1 | 2 | 5 | 1 | |||
Experimental sample | |||||||
Full stain | 1 | 2 | 5 | 1 | 5 | 2 |
Table 1: 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.
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 authors have nothing to disclose.
We would like to thank Tom Cheung (The Hong Kong University of Science & Technology) for advice on MuSC isolation. This work was funded by the NIAMS-IRP through NIH grants AR041126 and AR041164.
5 mL Polypropylene Round-Bottom Tube | Falcon | 352063 | |
5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap | Falcon | 352235 | |
20 G BD Needle 1 in. single use, sterile | BD Biosciences | 305175 | |
anti-Alpha 7 Integrin PE (clone:R2F2) (RatIgG2b) | The University of British Columbia | 53-0010-01 | |
APC anti-mouse CD31 Antibody | BioLegend | 102510 | |
APC anti-mouse CD45 Antibody | BioLegend | 103112 | |
BD FACSMelody Cell Sorter | BD Biosciences | ||
BD Luer-Lok tip control syringe, 10-mL | BD Biosciences | 309604 | |
Biotin anti-mouse CD106 Antibody | BioLegend | 105703 | |
C57BL/6J mouse (Female and Male) | The Jackson Laboratory | 000664 | |
B6.129S4-Pdgfratm11(EGFP)Sor/J mouse | The Jackson Laboratory | 007669 | |
Corning BioCoat Collagen I 6-well Clear Flat Bottom TC-treated Multiwell Plate | Corning | 356400 | |
Corning BioCoat Collagen I 12-well Clear Flat Bottom TC-treated Multiwell Plate | Corning | 356500 | |
Corning BioCoat Collagen I 24-well Clear Flat Bottom TC-treated Multiwell Plate | Corning | 356408 | |
DAPI Solution (1 mg/mL) | ThermoFisher Scientific | 62248 | |
Disposable Aspirating Pipets, Polystyrene, Sterile | VWR | 414004-265 | |
Donkey anti-Goat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | ThermoFisher Scientific | A-11055 | |
Falcon 40 µm Cell Strainer, Blue, Sterile | Corning | 352340 | |
Falcon 60 mm TC-treated Cell Culture Dish, Sterile | Corning | 353002 | |
Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 15-mL | VWR | 352196 | |
Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 50-mL | Corning | 352070 | |
Falcon Round-Bottom Tubes, Polypropylene, Corning | VWR | 60819-728 | |
Falcon Round-Bottom Tubes, Polystyrene, with 35um Cell Strainer Cap Corning | VWR | 21008-948 | |
Fibroblast Growth Factor, Basic, Human, Recombinant (rhFGF, Basic) | Promega | G5071 | |
FlowJo 10.8.1 | |||
Gibco Collagenase, Type II, powder | ThermoFisher Scientific | 17101015 | |
Gibco Dispase, powder | ThermoFisher Scientific | 17105041 | |
Gibco DMEM, high glucose, HEPES | ThermoFisher Scientific | 12430054 | |
Gibco Fetal Bovine Serum, certified, United States | ThermoFisher Scientific | 16000044 | |
Gibco Ham's F-10 Nutrient Mix | ThermoFisher Scientific | 11550043 | |
Gibco Horse Serum, New Zealand origin | ThermoFisher Scientific | 16050122 | |
Gibco PBS, pH 7.4 | ThermoFisher Scientific | 10010023 | |
Gibco PBS (10x), pH 7.4 | ThermoFisher Scientific | 70011044 | |
Gibco Penicillin-Streptomycin-Glutamine (100x) | ThermoFisher Scientific | 10378016 | |
Goat anti-Mouse IgG1 cross-absorbed secondary antibody, Alexa Fluor 555 | ThermoFisher Scientific | A-21127 | |
Hardened Fine Scissors | Fine Science Tools Inc | 14090-09 | |
Invitrogen 7-AAD (7-Aminoactinomycin D) | ThermoFisher Scientific | A1310 | |
Mouse PDGF R alpha Antibody | R&D Systems | AF1062 | |
Normal Donkey Serum | Fisher Scientific | NC9624464 | |
Normal Goat Serum | ThermoFisher Scientific | 31872 | |
Pacific Blue anti-mouse Ly-6A/E (Sca 1) Antibody | BioLegend | 108120 | |
Paraformaldehyde, 16% | Fisher Scientific | NCC0528893 | |
Pax7 mono-clonal mouse antibody (IgG1) (supernatant) | Developmental Study Hybridoma Bank | N/A | |
PE/Cyanine7 Streptavidin | BioLegend | 405206 | |
Student Vannas Spring Scissors | Fine Science Tools Inc | 91500-09 | |
Student Dumont #5 Forceps | Fine Science Tools Inc | 91150-20 | |
Triton X-100 | Sigma-Aldrich | T8787 |