Protocol for the isolation of mouse fibro/adipogenic progenitors using magnetic-activated cell sorting

Summary

Fibro/adipogenic progenitors (FAPs) are mesenchymal progenitors that support muscle stem cell activation and modulate the extracellular matrix to facilitate muscle regeneration. Here, we present a protocol for the isolation of FAPs from mouse skeletal muscle. We describe steps for performing enzymatic digestion, mechanical dissociation, and magnetic-activated cell sorting (MACS). This protocol enables the isolation of FAPs with high yield and purity, facilitating research on muscle disorders and advancing muscle regeneration strategies.

Graphical abstract

Highlights

• •Instructions for skeletal muscle isolation and dissociation to obtain FAPs
• •Guidance on gentle magnetic-activated cell sorting to minimize cell damage
• •Procedure for evaluating FAPs’ purity and culturing and isolating FAPs-derived EVs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Fibro/adipogenic progenitors (FAPs) are mesenchymal progenitors that support muscle stem cell activation and modulate the extracellular matrix to facilitate muscle regeneration. Here, we present a protocol for the isolation of FAPs from mouse skeletal muscle. We describe steps for performing enzymatic digestion, mechanical dissociation, and magnetic-activated cell sorting (MACS). This protocol enables the isolation of FAPs with high yield and purity, facilitating research on muscle disorders and advancing muscle regeneration strategies.

Before you begin

FAPs play a pivotal role in muscle regeneration and hemostasis.¹ Following muscle acute injury, FAPs secrete cytokines and growth factors that promote the activation and differentiation of muscle stem cells (satellite cells), while also modulating immune response to facilitate efficient tissue repair.^1,2 Furthermore, FAPs are essential in remodeling the extracellular matrix (ECM), providing the necessary structural and environmental support required for optimal muscle function and repair.¹ However, under pathogenic conditions, FAPs can differentiate into adipocytes and fibroblasts, contributing to fat infiltration and fibrosis in muscle disorders and aging.^3,4 These dual roles of FAPs-supporting regeneration under normal conditions but contributing to degeneration in disease states-highlight the need for a deeper understanding of their molecular and functional properties. To address this, we present a protocol for the isolation of mouse FAPs through MACS from wild-type mice. This approach has also been successfully applied to isolating FAPs from acutely injured skeletal muscle. Given the importance of FAPs in muscle regeneration and degeneration, this refined FAP isolation method will contribute to advancing research on muscle disorders and facilitate the development of improved strategies for effective muscle regeneration and repair.

Institutional permissions

All animal experiments described in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Georgia. Others who plan to replicate this protocol should receive appropriate approval from their institution.

Preparation of muscle digestion media, FAP growth media, and gelatin-coated plate

Timing: ∼15 min

• 1.Freshly prepare Digest 1 mix and Digest 2 mix, keep them on ice prior to use. Prepare PBS for collecting dissected skeletal muscle and keep it on ice.
• 2.Freshly prepare the growth media for culturing FAPs. Prewarm the media in 37°C water bath for 5–10 min just before use.
• 3.Precoat wells for cell culture with enough gelatin to cover the entire bottom of the well and incubate the plates for approximately 2–3 h in a 37°C incubator before seeding isolated cells.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CD45-biotin, mouse, 1: 50	Miltenyi Biotec	Cat#130-124-209
CD31-biotin, mouse, 1:50	Miltenyi Biotec	Cat#130-119-662
Anti-integrin alpha 7-biotin, mouse, 1:50	Miltenyi Biotec	Cat#130-128-938
Anti-biotin MicroBeads, 1:5	Miltenyi Biotec	Cat#130-090-485
Anti-mPDGFRα, 1:200 (5 μg/mL)	R&D Systems	Cat#AF1062
Anti-mouse Ly-6A/E, 1:100	BD Biosciences	Cat#557403
Anti-mouse myosin heavy chain, 1:200 (5 μg/mL)	DSHB	MF 20
Chemicals, peptides, and recombinant proteins
Human basic fibroblast growth factor (bFGF)	EMD Millipore	Cat#GF003
Dulbecco’s modified Eagle’s medium (DMEM), high glucose	Gibco	Cat#11-995-065
Collagenase type II, powder	Worthington Biochem	Cat#LS004176 (1 gm)
Collagenase/dispase, powder	MilliporeSigma	Cat#10269638001
Fetal bovine serum (FBS)	Neuromics	Cat#FBS002
Ham’s F-10 nutrient mix (F10)	Gibco	Cat#11550043
Gelatin, 0.1%	STEMCELL Technologies	Cat#07903
Phosphate-buffered saline (PBS), without calcium and magnesium	Corning	Cat#21-040-CV
Triton X-100	Thermo Fisher Scientific	Cat#J66624.AP
Penicillin-streptomycin (P-S), 10,000 U/mL	Gibco	Cat#15140122
Trypan blue	Corning	Cat#25-900-CI
Experimental models: Organisms/strains
Mouse: C57BL/6J, male, approximately 8 weeks old	The Jackson Laboratory	Cat#000664
Other
40 μm cell strainers, sterile	Corning	Cat#07-201-430
Glass Pasteur pipette	Fisher Scientific	Cat#13-678-20D
5 mL syringe	Fisher Scientific	Cat#14955452

Materials and equipment

• •Wash Solution (WS)

Reagent	Final concentration	Amount
FBS	10%	5 mL
Penicillin/Streptomycin (P-S), 10,000 U/mL	1%	500 μL
F10	N/A	44.5 mL
Total	N/A	50 mL

Keep on ice, prepare fresh media every time.

• •Digest 1 mix: WS with 2.433 mg/mL Collagenase Type II. [Keep on ice, prepare fresh media every time]
• •Digest 2 mix: WS with 2.5 mg/mL Collagenase/Dispase. [Keep on ice, prepare fresh media every time].
• •Plate coating medium: Gelatin
• • Growth Media (GM)

Reagent	Final concentration	Amount
Heat-inactivated Fetal Bovine Serum (FBS)	20%	4 mL
Human basic fibroblast growth factor (bFGF) (50 μg/mL aliquot)	2.5 ng/mL	1 μL
Penicillin/Streptomycin (P-S), 10,000 U/mL	1%	200 μL
Dulbecco’s modified Eagle’s medium (DMEM), high glucose	N/A	15.8 mL
Total	N/A	20 mL

Keep on ice, prepare fresh media every time, and pre-warm in a 37°C water bath for 5–10 min before use.

Step-by-step method details

Extraction of mice skeletal muscle

Timing: ∼15 min per mouse

This step isolates skeletal muscle from euthanized mice to obtain FAPs.

• 1.
  Sanitization and Setup:

  • a.
    Clean and sanitize the dissection area thoroughly to maintain a sterile environment with 70% ethanol.
  • b.
    Arrange all the necessary dissection tools (e.g., scissors, forceps, scalpel) and have them sterilized.
• 2.
  Confirmation of Euthanasia:

  • a.
    Euthanize mice according to your approved IACUC protocol.
• 3.
  Positioning the Mouse:

  • a.
    Lay the mouse on its back on a clean, sterile surface.

Note: To stabilize the body, you can pin down the limbs using needles or similar tools designed for dissection purposes. This immobilization is crucial for precise incisions and manipulations during the dissection process.

• 4.
  Skin Incision and Exposure:

  • a.
    Spray down mouse limb with 70% ethanol (Figure 1A).
  • b.
    Make a transverse incision at the terminal part of the hindlimb using sterilized scissors or a scalpel and carefully peel back the skin to expose the underlying skeletal muscle tissue (Figures 1B and 1C).

Note: It's important to make this initial incision as clean and precise as possible to facilitate easy access to the muscle tissue while minimizing potential damage to the underlying structures.

Note: Additionally, ensure minimal to no hair comes into contact with the exposed muscle to reduce the risk of contamination.

Note: Handle sharp instruments with care to prevent injury.

• 5.
  Dissection of Skeletal Muscles:

  • a.
    Carefully remove the fascia on top of the tibialis anterior (TA) muscle with micro-dissection tweezer.
  • b.
    Gently isolate the skeletal muscle tissue, such as extensor digitalis longus, soleus, and gastrocnemius (GAS), avoiding pulling too forcefully on the muscle during removal from the hindlimb (Figures 1D–1F).

    Note: Carefully remove surrounding connective tissues, such as tendons, without damaging the muscles to ensure sample purity and to maximize the yield of viable FAPs.

  • c.
    Place dissected skeletal muscles into the pre-cold PBS.

Figure 1.

Extraction of hindlimb skeletal muscles from adult mice

Mouse skeletal muscle dissection.

(A) Spray the hindlimb of the mice with 70% ethanol.

(B and C) Make a transverse incision along the terminal part of the hindlimb and peel back the skin to expose the skeletal muscle.

(D–F) Dissect the TA and GAS muscles.

Enzymatic and mechanical dissociation of skeletal muscle to release mononuclear muscle cells

Timing: ∼2 h for 1 mouse; add 15–30 min for each additional mouse

This step releases mononuclear cells from muscle for single-cell sorting through mechanical dissociation and enzymatic digestion.

• 6.
  Mince Muscle Tissue in the Hood:

  • a.
    Place the isolated muscle tissues on a sterile plate inside the hood and rinse them twice with PBS to remove any remaining fur from their surface (Figure 2A).
  • b.
    Mince the muscle tissue using sterile scissors into small pieces, approximately 2–3 mm³ in size (Figure 2B).
• 7.
  First Digestion Step:

  • a.
    Transfer the minced muscle into the Digest 1 Mix solution, using 1 mL per GAS and 500 μL per TA (Figure 2C).
  • b.
    Seal the tube with parafilm to prevent leakage and contamination.
  • c.
    Incubate the tubes in a 37°C water bath for 30 min. Vortex gently after 15 min to enhance digestion efficiency (Figure 2D).
  • d.
    Centrifuge at 150 × g for 5 min at 4°C (Figure 2E).
  • e.
    Transfer the supernatant to a 15 mL tube, add an equal volume of pre-cooled Wash Solution (WS), and keep it on ice for subsequent filtration.
• 8.
  Second Digestion Step:

  • a.
    Resuspend the pellet from the first centrifugation in Digest 2 Mix using 1 mL per GAS and 500 μL per TA, seal the tube with parafilm, and incubate in a 37°C water bath for 15 min (Figure 2F).
  • b.
    Following incubation, dilute the mixture 1:1 with WS, then gently mince it three times using a 5 mL syringe equipped with an 18G needle to further dissociate the tissue (Figure 2G).
  • c.
    Filter the suspension through a 40 μm cell strainer into a 50 mL tube, combining it with the previously set aside supernatant.
• 9.
  Final Centrifugation and Cell Resuspension:

  • a.
    Centrifuge the combined filtrate at 150 × g for 5 min at 4°C.
  • b.
    Carefully aspirate the supernatant, ensuring minimal disturbance to the cell pellet.
  • c.
    Resuspend the cell pellet in WS (10⁶ cells per 50 μL) for downstream applications.

Figure 2.

Mechanical and enzymatic dissociation of skeletal muscle to release single cells

Trituration of skeletal muscle TA and GAS to release myofibers.

(A) Wash the muscles with PBS to remove any remaining fur on the muscles.

(B) Mince the muscle with scissors into pieces of approximately 2–3 mm³ in size.

(C) Transfer the minced muscles into Digest 1 mix solution in a 15 mL tube and seal it.

(D) Transfer the tube into a 37°C water bath.

(E) Minced muscle incubated in Digest 1 for 30 min at 37°C in water bath.

(F) After a 15-min incubation in Digest 2 within a water bath (37°C).

(G) Gently mince the tissue mixture by passing through a 5 mL syringe fitted with an 18G needle three times.

Antibody staining of mono-nuclear cells associated with skeletal muscle

Timing: ∼1 h for 1 mouse; add 15–30 min for each additional mouse

This step isolates FAPs from mononuclear cells released from skeletal muscle by incubating them with specific antibodies to negatively exclude hematopoietic cells (anti-CD45 antibody), endothelial cells (anti-CD31 antibody), and muscle satellite cells (anti-integrin alpha 7 antibody), and sort the target population.

• 10.Label MACS tubes (1.5 mL tube) for each experimental sample.
• 11.Transfer the cell suspension from the 50 mL tube into their respective MACS tubes.
• 12.Stain cell samples with biotin-conjugated anti-CD31, CD45, integrin alpha 7 antibodies at a 1:50 dilution for 15 min in a 4°C refrigerator.
• 13.Centrifuge the cells at 150 × g for 5 min at room temperature (RT).
• 14.Aspirate the supernatant and resuspend the cell pellet in WS (10⁶ cells per 50 μL), gently pipetting up and down to fully dissociate into a single-cell suspension.
• 15.Add Anti-Biotin microbeads at a 1:5 dilution and incubate for 15 min in a 4°C refrigerator.
• 16.While incubating, prepare the magnetic and rinsing columns by rinsing each column with 3 mL of WS (Figure 3).
• 17.Transfer the cell suspension into the column and collect the flow-through containing unlabeled cells.
• 18.Wash the column twice with 1 mL of WS, collecting the pass-through cells and combining them with the previous flow-through.
• 19.Centrifuge the combined cells at 150 × g for 4 min at RT.
• 20.During centrifugation, aspirate gelatin from the coating plate, rinse the plate with PBS and add FAPs pre-warmed culture medium.
• 21.After centrifugation, carefully aspirate the supernatant and resuspend the cell pellet with 1 mL FAPs culture medium.
• 22.Determine the cell concentration using a cell counter.
• 23.Seed the cells at a density of 10,000 per cm² in wells pre-filled with FAP cultural medium. Change the medium every other day until cells reach 80% confluency.

Figure 3.

MACS isolation of FAPs

The mixed cells flow through the MACS column, with PDGFRα⁺ cells being sorted out after negative selection using anti-Biotin microbeads, which bind to Biotin-conjugated cells.

Downstream ex vivo analysis of sorted FAPs

In this section we discuss downstream applications including cell culture, immunofluorescence (IF), isolation of FAPs derived extracellular vesicles.

• 24.
  Passaging of FAPs:

  • a.
    Precoat wells for cell culture with gelatin 2–3 h prior to seeding cells.
  • b.
    Remove the growth medium (GM) and rinse the cells with PBS.
  • c.
    Add warmed Accutase to cover the cells and incubate wells for 5 min at 37°C.
  • d.
    Confirm under a microscope that >90% of the cells have detached from the well.
  • e.
    Gently add an equal volume of warmed GM to dilute the Accutase and transfer the cell suspension into a 15 mL tube.
  • f.
    Centrifuge the cell suspension at 300 × g for 4 min at RT.
  • g.
    Aspirate the supernatant and resuspend the cell pellet in GM for cell counting.
  • h.
    Seed cells at the appropriate density (such as 10,000 per cm²) and continue culturing in GM.

Note: Passage the isolated FAPs when they reach approximately 80% confluent to prevent differentiation. After 3–4 days of culturing, the isolated FAPs are usually ready for passage.

Refresh the GM every other day to maintain optimal conditions.

• 25.
  Immunofluorescence staining of the FAPs:

  • a.
    Remove the GM from each well. Rinse the cells with PBS to remove any residual GM.
  • b.
    Add enough 4% paraformaldehyde (PFA) to cover the cells and incubate at RT for 15 min to fix them.
  • c.
    Remove the 4% PFA solution and wash the cells three times with PBS, allowing 5 min for each wash.
  • d.
    Permeabilize cells with 0.1% Triton-X solution for 10 min at RT.
  • e.
    Wash the cells with PBS three times to remove the Triton-x solution.
  • f.
    Add blocking buffer (5% Normal Donkey Serum, 5% BSA in PBS) for 1 h at RT to block non-specific binding sites.
  • g.
    Aspirate the blocking buffer, add the primary antibody diluted in blocking buffer to each well and incubate overnight (∼12 h) at 4°C.
  • h.
    Remove the primary antibody solution and wash the cells four times for 15 min with PBS at RT to ensure thorough removal of unbound primary antibody.
  • i.
    Add the secondary antibody diluted in PBS to cover the cells and incubate for 1 h at RT.
  • j.
    Remove the secondary antibody solution from the well and wash the cells with PBS our times for 15 min each at RT.
  • k.
    Add DAPI solution (1 μg/m DAPI in PBS) to the cells and incubate for 5 min at RT to stain the nuclei.
  • l.
    Remove the DAPI staining solution and wash the cells with PBS for 5 min to remove excess DAPI.
  • m.
    After the final wash, the cells are ready for imaging. Cover the wells to prevent any contamination or drying out of the cells.
• 26.
  Collect extracellular vesicles (EVs) derived from the FAPs:

  • a.
    Culture FAPs in GM until they reach 70–80% confluency.
  • b.
    Replace the GM without FBS to avoid contamination with exogenous vesicles from the serum and incubate for 24 h to allow the cells to release EVs into the media.
  • c.
    After a 24-h incubation, collect the conditioned media from the cell culture and refeed the cells with GM without FBS, and allow the cells to incubate for another 24 h. Collect the conditioned medium again after the second 24-h incubation period, resulting in two separate collections of conditioned medium.
  • d.
    Centrifuge the collected media at 300 × g for 10 min to remove cells and large debris.
  • e.
    Transfer the supernatant to a new tube and centrifuge again at 1,000 × g for 30 min to remove smaller debris and apoptotic bodies.
  • f.
    Transfer the supernatant to ultracentrifuge tubes.
  • g.
    Ultracentrifuge at 100,000 × g for 2 h at 4°C to pellet the EVs.
  • h.
    Carefully remove the supernatant without disturbing the EV pellet.
  • i.
    Washing the EV pellet in pre-cold PBS.
  • j.
    Ultracentrifuge again at 100,000 × g for 1 h at 4°C to pellet the EVs.
  • k.
    Remove the supernatant and resuspend the EV pellet in a small volume of PBS.
  • l.
    Store the isolated EVs at −80°C for long-term storage. Avoid repeated freeze-thaw cycles.

Expected outcomes

After completing the detailed isolation procedure outlined above, muscle-resident endothelial cells, hematopoietic cells, and muscle satellite cells are effectively removed through negative selection, while FAPs are enriched through positive selection. Utilizing a combination of two-step enzymatic and mechanical dissociation of skeletal muscle along with the MACS protocol, we typically obtain approximately 600,000 FAPs from two TA and two GAS muscles from healthy adult mice aged 12–16 weeks. FAPs were immunostained for PDGFRα, a cytoplasmic marker specific to FAPs, immediately after MACS isolation (Figure 4A). Despite the sorted cells had not fully spread at this stage, immunostaining confirmed their high purity. After three days in culture, the isolated FAPs typically reached confluency. Their identity and purity were further validated by immunostaining for PDGFRα and Myosin Heavy Chain (MHC), with the latter serving as a marker for muscle myofibers (Figure 4B). Furthermore, co-immunofluorescence staining for PDGFRα and Sca1 demonstrated a purity level exceeding 90% (Figure 4C).

Figure 4.

Immunofluorescent staining of MACS-isolated FAPs

(A) Immediately after MACS, FAPs were immunostained for nuclei (DAPI, blue) and PDGFRα (green). Scale bar: 70 μm.

(B) Two days after culture, FAPs were immunostained for nuclei (DAPI, blue), PDGFRα (green), and MHC (red). Scale bar: 70 μm.

(C) Two days after culture, FAPs were immunostained for nuclei (DAPI, blue), PDGFRα (green), and Sca1 (red). Scale bar: 130 μm.

Limitations

This method predominantly yields FAPs, however, the isolation process may alter their cellular status. Additionally, ex vivo culturing could further modify FAP characteristics. It is crucial to acknowledge that both the isolation process and subsequent ex vivo culturing can potentially affect the intrinsic properties of FAPs. Further studies are necessary to investigate these effects, as they are essential for refining the isolation technique and ensuring the reliability and validity of subsequent experimental outcomes.

Troubleshooting

Problem 1

Low purity of isolated FAPs.

Potential solution

• •Gently resuspend cell pellets thoroughly before adding biotin-conjugated antibodies and Anti-Biotin microbeads to make sure they can access all the single cells. Additionally, optimizing dilution of these antibodies and microbeads according to the cell amounts specified in the product sheet. Finally, apply the appropriate column based on the number of cells to isolate them effectively.
• •Add the FcR Blocking Reagent (Miltenyi Biotec, Cat# 130-092-575) to the wash buffer when resuspending cell pellets collected from muscle tissue. Mix the buffer with the cells and incubate for 10 min in a 4°C refrigerator. Subsequently, incubate the cells with biotin-conjugated antibodies. This step may enhance the specificity of labeling with MACS antibodies or MACS microbeads, improving the purity of isolated FAPs.

Problem 2

Low yield of MACS-sorted FAPs.

Potential solution

• •Increase the quantity of extracted skeletal muscle. Carefully and thoroughly dissect the skeletal muscles of interest to maximize cell yield.
• •Avoid over-digestion of skeletal muscle, be cautious with digestion times. Over-digestion during incubation in Digest mix 1 and 2 can potentially decrease the viability of the cells. Optimize digestion times to balance between effective tissue dissociation and cell viability

Problem 3

Too many blood cells in the final isolated cells.

Potential solution

• •Resuspend the cell pellet in 1 mL of Red Blood Cell Lysis solution to remove erythrocytes after isolating the cells from muscle tissue. Incubate at room temperature for no more than 2 min. Centrifuge at 150 × g for 10 min and aspirate the supernatant completely. Next, resuspend the cells in an appropriate amount of wash medium containing biotin-conjugated antibodies, continuing step 12.

Problem 4

Primary FAPs do not attach well after first passage.

Potential solution

• •To detach FAPs from the plate, apply Accutase instead of Trypsin to incubate the cells at 37°C for 5 min. Gently tap the plate to ensure the cells are fully detached. Dilute the accutase-cell mixture with freshly prepared pre-warmed culture medium, then collect the cell pellets by centrifuging at 300 × g for 4 min. Gently resuspend the cell pellets in the pre-warmed culture medium and seed them onto a gelatin-coated plate.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yao Yao (Yao.Yao@uga.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Jinghui Gao (jinghui.gao@uga.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

No data or code was generated by this study.

Acknowledgments

This research was partially funded by startup funds from the University of Georgia (to Y.Y.) and the Department of Defense Congressionally Directed Medical Research Programs Amyotrophic Lateral Sclerosis Research Program Therapeutic Idea Award (W81XWH2210261 to Y.Y.). We would like to thank Dr. Hyojung Choo (Emory University) for sharing valuable experience related to MACS.

Author contributions

Conceptualization, J.G. and Y.Y.; methodology, J.G., Y.Z., and A.S.; writing, all authors; supervision and funding acquisition, Y.Y.

Declaration of interests

The authors declare no competing interests.