Protocol to assess myogenic effects of 3T3-L1 and primary murine fibro-adipogenic progenitors on C2C12 myoblast differentiation

Summary

Fibro-adipogenic progenitors (FAPs) are key regulators of skeletal muscle regeneration and influence myogenic differentiation. Here, we present a protocol for the isolation of primary FAPs from injured murine skeletal muscle and the co-culture of C2C12 myoblasts with either 3T3-L1 or primary FAPs. We describe steps for muscle injury, harvest, and digestion followed by FAP isolation. We then detail procedures for co-culture and for assessing myogenic differentiation using immunofluorescence imaging, enabling direct comparison of stromal influences on myoblast differentiation.

For complete details on the use and execution of this protocol, please refer to Norris et al.¹

Graphical abstract

Highlights

• •Isolate primary fibro-adipogenic progenitors from injured skeletal muscle
• •Co-culture C2C12 myoblasts with either 3T3-L1 preadipocytes or primary FAPs
• •Compare effects of 3T3-L1 and primary FAP population on myoblast differentiation
• •Quantify myoblast differentiation using immunofluorescence imaging

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

Fibro-adipogenic progenitors (FAPs) are key regulators of skeletal muscle regeneration and influence myogenic differentiation. Here, we present a protocol for the isolation of primary FAPs from injured murine skeletal muscle and the co-culture of C2C12 myoblasts with either 3T3-L1 or primary FAPs. We describe steps for muscle injury, harvest, and digestion followed by FAP isolation. We then detail procedures for co-culture and for assessing myogenic differentiation using immunofluorescence imaging, enabling direct comparison of stromal influences on myoblast differentiation.

Before you begin

Skeletal muscle regeneration requires coordination between multiple cell types, including muscle stem cells (MuSCs), immune cells, endothelial cells, peripheral nerve-associated cells, and fibro-adipogenic progenitors (FAPs) (reviewed in^2,3,4,5,6). Due to this complexity, it is difficult to dissect the direct interactions between FAPs and myogenic cells to ensure proper regeneration in vivo.

The 3T3-L1 cell line, derived from mouse embryonic fibroblasts, is a well-established preadipocyte model.⁷ It is widely used due to its ease of culture, genetic stability, and reproducible adipogenic differentiation.^8,9,10,11,12 As such, it provides a robust system to explore adipogenic influences on myoblast differentiation in vitro. However, recent single-cell RNA sequencing studies have revealed that primary FAPs represent a heterogeneous population within skeletal muscle.^{13,14,15,16,17,18,19} While they all commonly express PDGFRα, FAP subpopulations can be distinguished by tissue localization and transcriptional signatures, suggesting distinct roles during regeneration.^{13,20,21,22,23,24} This heterogeneity underscores the importance of validating in vitro findings from uniform cell lines with heterogenous primary FAPs derived from murine skeletal muscle.

This protocol describes the isolation of primary FAPs from injured murine skeletal muscle and explains how to co-culture the myoblast line C2C12 with either primary FAPs or 3T3-L1 cells, enabling direct comparison of how each stromal population influences myogenic differentiation. We also provide guidance on analyzing myogenic differentiation outcomes through immunofluorescence following co-culture with either primary FAPs or 3T3-L1 cells.

Before proceeding with this protocol, readers must seek approval from IACUC for this experiment and conform to the relevant regulatory standards.

Innovation

FAPs are key regulators of skeletal muscle regeneration but exhibit substantial heterogeneity in vivo. Conventional in vitro studies often rely on the 3T3-L1 preadipocyte cell line, which offers consistency and rapid results but fails to reflect in vivo FAP complexity. This protocol establishes a framework to validate 3T3-L1 findings by directly comparing them with primary FAPs isolated from injured skeletal muscle using differential plating. By integrating established fibroblast enrichment through differential plating, with a standardized co-culture platform, this workflow enables systemic evaluation of how heterogenous primary FAP populations influence myoblast differentiation. This approach bridges the gap between high throughput cell line models and physiologically relevant primary cells, providing a practical tool to study stromal-myogenic interactions during regeneration.

Institutional permissions

Adult 9-13 weeks old 129S1/SvlmJ mice of both sexes were housed in standard ventilated cages at a controlled temperature (22°C–23°C), with 40%–50% humidity and ad libitum access to food and water. All animal work was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida. Readers must acquire permission from relevant institutions before proceeding.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Chicken anti-Myosin heavy chain (MHC) (1:50)	DSHB	Cat #: MF 20; RRID: AB_2147781
Alexa Fluor 488 Donkey anti-chicken (1:1000)	Invitrogen	Cat #: A78948; RRID: AB_2921070
DAPI	Invitrogen	Cat #: D1306
Chemicals, peptides, and recombinant proteins
Dulbecco’s Modified Eagle’s Medium (DMEM)	Gibco	Cat #: 11965-092
Fetal Bovine Serum (FBS)	Invitrogen	Cat #: 10438026
GlutaMax	Gibco	Cat #: 35050-061
Horse serum (HS)	Gibco	Cat #: 26050-070
Newborn calf serum (NCS)	Gibco	Cat #: 16010-159
Glycerol (GLY)	Acros Organics	Cat #: 56-81-5
Cardiotoxin (CTX)	Lotaxan	Cat #: L8102-1MG
Collagenase IV	Worthington	Cat #: LS004188
Dispase	Gibco	Cat #: 17105041
Insulin	Sigma	Cat #: I2643
Human fibroblast growth factor basic (FGFb)	Fisher Scientific	Cat #: PHG0264
Troglitazone	Sigma	Cat #: T2573
Experimental models: Cell lines
Mouse: C2C12	ATCC	Cat #: CRL-1772; RRID: CVCL_0188
Mouse: 3T3-L1	Zenbio	Cat #: SP-L1-F
Experimental models: Organisms/strains
Mouse: 129S1/SvlmJ Adult 9-13 weeks of both sexes	Jackson Laboratories	Strain #: 002448; RRID: IMSR_JAX:002448
Software and algorithms
Fiji (ImageJ)	N/A	RRID: SCR_002285
Other
70μm Cell Strainer	Fisher Scientific	Cat #: 22-363-548
100μm Cell Strainer	Fisher Scientific	Cat #: 22-363-549
Tissue culture-treated dish 100mm	STEMCELL Technologies	Cat #: 100--0082
Cell culture flask, T-75 cm2	STEMCELL Technologies	Cat #: 353135

Materials and equipment

Wash Buffer

Reagent	Final concentration	Volume
Ham’s F10 Nutrient Mix	N/A	27mL
Inactivated FBS	10%	3mL
Total	N/A	30mL

Wash buffer can be prepared in advance, stored at 4°C and used up to a month after.

Digestion Buffer

Reagent	Final concentration	Volume
DMEM	N/A	32.5mL
Collagenase II	0.15%	6mL from 1% stock
Dispase II	0.04%	1.6mL from 1% stock
Penicillin & Streptomycin	1%	400uL
CaCl2	4.3mM	4.8uL from 2.5M stock
Total	N/A	45.3mL

Prepare fresh on the day of isolation and keep on ice to prevent enzyme activation.

Alternatives: Wash Buffer can be used instead of DMEM.

Fibroblast Cell Culture Media (Primary FAPs and 3T3-L1 cells)

Reagent	Final concentration	Volume
DMEM	N/A	44.5mL
Newborn Calf Serum (NCS)	10%	5mL
Glutamax	1%	500uL
Total	N/A	50mL

Media can be aliquoted in 50mL tubes and stored at −20°C for up to 6 months.

Cell Culture Media for C2C12 maintenance

Reagent	Final concentration	Volume
DMEM	N/A	44.5mL
Fetal Bovine Serum (FBS)	10%	5mL
Glutamax	1%	500uL
Total	N/A	50mL

Media can be aliquoted in 50mL tubes and stored at −20°C for up to 6 months.

Cell Culture Media for C2C12 differentiation

Reagent	Final concentration	Volume
DMEM	N/A	44mL
Glutamax	1%	500uL
Horse Serum (HS)	2%	1mL
Total	N/A	50mL

Media can be aliquoted in 50mL tubes and stored at −20°C for up to 6 months.

Blocking Solution

Reagent	Final concentration	Volume
10x PBS	1X	5mL
MiliQ Water	N/A	40.75mL
Donkey Serum	2%	2.5mL
Triton X-100	0.3%	750uL of 20% stock
Sodium Azide	0.2%	1mL of 10% stock solution
Total	N/A	50mL

Once prepared, use within a month and store at 4°C.

PBS-T

Reagent	Final concentration	Volume
10x PBS	1X	100mL
MiliQ Water	N/A	900mL
Tween-20	0.1%	1mL
Total	N/A	1L

Once prepared, solution can be used within 2-3 months and kept at 20°C–25°C.

Step-by-step method details

Muscle injury and harvest

Timing: 3 days

In this step, we describe how to perform muscle injuries in adult mice and how to harvest before proceeding to tissue processing and FAP isolation. For a step-by-step protocol and instructional video, refer to Connor et al.²⁵

Note: The following steps should be followed according to IACUC guidelines.

• 1.Anesthetize mice with 1.5%–2% isoflurane and 2% oxygen.
• 2.Gently wipe the Tibialis Anterior (TA) with an alcohol wipe.
• 3.Inject 40–50uL of 10mM Cardiotoxin (CTX) or 50% Glycerol (GLY).

Note: Smaller mice will require less compared to larger, often male mice.

• 4.Allow mice to recover for 3 days and euthanize through overdose of isoflurane, or according to approved laboratory’s IACUC procedures.
• 5.When dissecting TAs, maintain all dissecting tools in a small beaker with 70% ethanol to minimize contamination and thoroughly spray the lower half of the mouse with 70% ethanol.

CRITICAL: A significant source of contamination comes from fur. Therefore, heavily spray the animal and maintain dissecting tools submerged in 70% ethanol when not in use.

• 6.Dissect TAs and in two separate cell culture plates on ice, wash twice with ice cold PBS and 1% penicillin and streptomycin.

Isolation of primary FAPs through differential plating

Timing: 2.5 h

In this step we describe mechanical and enzymatic digestion of harvested muscles, followed by isolation of FAPs through differential plating. We recommend 3–4 TAs, or 1 TA and 1 Gastrocnemius, per sample to provide enough cells. Keep all buffers on ice until use.

• 7.
  With TAs on ice, add 500 uL of Wash buffer and mechanically mince muscles (Figure 1).

  • a.
    Begin by holding the TA by the tendon with forceps and with a scalpel finely cut along the grain of the muscle until most tissue is disassociated.
  • b.
    With dissecting scissors, proceed to cut tissue into fine pieces, ideally until pieces are 1-3mm in diameter.

Note: This must take roughly 5–7 min, avoid taking too long in this step. It is intended to facilitate enzymatic digestion.

• 8.
  Once mechanically minced, proceed to enzymatically digest muscle.

  • a.
    With dissecting forceps, move larger tissue pieces into a 50ml conical tube. Add 4.5mL of Digestion buffer to the plate with remaining muscle digestion.

CRITICAL: Maintain digestion solution on ice to prevent enzyme activation.

• 9.Place 50mL conical tube with muscle slurry and digestion mix in a shaking incubator set at 37°C at roughly 45 degrees to allow maximal movement of the solution within the conical tube.
• 10.Every 10–15 min remove sample and vortex for roughly 20 seconds and place back into incubator. Repeat this for 30–45 min until solution is mostly homogenous.

Note: The amount of time for digestion will depend on many factors like degree of injury to tissue, age of mouse, and amount of fibrosis. Proceed to troubleshooting 1 for more information.

• 11.After digestion, all steps should be carried out under sterile conditions.
• 12.In cell culture hood, place a 70 μm filter in a 50 mL conical tube. Troubleshooting 2 for more information.
• 13.Wet filter with 2 mL of Wash buffer.

CRITICAL: Wetting the filter prevents cells from strongly adhering to the filter, ensuring proper filtration.

• 14.Add digestion solution and allow solution to filter through.
• 15.Add 5 mL of Wash Buffer through filter to facilitate filtration and remaining cells through.
• 16.Centrifuge filtrate at 300 x g for 5 min.
• 17.Discard supernatant and resuspend in 5 mL of warmed Fibroblast cell culture media.
• 18.Plate suspension onto a 100mm tissue culture dish (P100) or T-75 tissue culture flask (T75).
• 19.Incubate at 37°C with 5% C0₂ for 30–40 min to allow fibroblasts to adhere to plate.
• 20.Remove supernatant and gently wash twice with warmed PBS. Proceed to troubleshooting 3 for more information.

Note: Pipet warm PBS on the side of the plate and gently rotate to wash. Avoid pipetting directly on top of cells to reduce washing off cells.

• 21.Add enough warmed Fibroblast cell culture media to generously cover the cells.
• 22.Allow 2 days for cells to expand until ∼80% confluency and utilize in step 26. Proceed to troubleshooting 4 and troubleshooting 5 for more information.

Pause point: Isolated cells can be frozen and stored until required for experiments.

Figure 1.

Mechanic and enzymatic digestion of injured muscles

3T3-L1 and primary FAP co-culture with C2C12

Timing: 10 days

In this step we describe how to test the effect of fibroblasts, primary FAPs or 3T3-L1 cells, on myoblast differentiation through co-culture.

• 23.Thaw a vial of C2C12 cells in P100/T75 and allow them to expand for 3 days with C2C12 maintenance media.
• 24.Two days after C2C12 thaw, thaw 3T3-L1 cells in a separate P100/T75 and allow to expand in fibroblast cell culture media for 3 days until ∼80% confluency.
• 25.
  After 3-day expansion of C2C12 cells, plate 1 x 10⁴ C2C12 cells per 24-well plate (or 526 cells/mm²).

  • a.
    Aspirate cell culture media.
  • b.
    Gently wash cells with warm PBS.
  • c.
    Add enough trypsin to cover cells and incubate at 37°C for 3min.
  • d.
    Add 6 mL C2C12 maintenance media to stop reaction.
  • e.
    Centrifuge at 300 x g for 5min at 20°C–25°C.
  • f.
    Aspirate supernatant and resuspend in 6 mL of C2C12 cell culture media.
  • g.
    Calculate cell density through hemocytometer or Burker camera.
• 26.
  After 2 days from C2C12 plating onto 24-well plate, cells are confluent and ready for differentiation with fibroblasts.

  • a.
    Obtain fibroblast suspension following step 22 and resuspend cell pellet in C2C12 differentiation media.
  • b.
    Plate 1 x 10⁴ 3T3-L1 cells or primary FAPs per 24-well plate (or 526 cells/mm²) on top of previously seeded C2C12s.

CRITICAL: C2C12s must be confluent to ensure proper and reproducible differentiation.

• 27.Allow C2C12 cells to differentiate for 5 days, changing media every other day with C2C12 differentiation media.

Optional: To investigate indirect effects between FAPs and C2C12, collect the supernatant of primary FAPs/3T3-L1 and mix with C2C12 media at 1:1 or 1:2 ratios and change media every 24 h with fresh supernatant/media mix.

• 28.After 5 days, remove media, wash with warm PBS twice and add 2mL of ice-cold 4% PFA for 2 hrs.

Immunofluorescence and image analysis

Timing: 3 days

In this step we describe how to determine myogenic outcomes such as fusion index, the number of nuclei fused to form myotubes, and differentiation index, the amount of myotubes formed. For this, mature myotubes are visualized through the antibody MF-20 and all nuclei through DAPI.

• 29.Following PFA fixation, wash cells with cold PBS-T for 5min 3 times.
• 30.Add 200–300uL of blocking solution per 24-well for 2hrs at 4°C.
• 31.Prepare MF-20s antibody dilution at 1:50 in blocking solution and incubate 16-20hrs at 4°C. Add 200-300uL of antibody solution per 24-well plate.

Note: Alternatively, you can incubate for 2 h at 20°C–25°C.

• 32.Remove antibody solution and gently wash 3 times for 5min with PBS-T at 20°C–25°C.
• 33.Prepare secondary antibody dilution by adding mouse-Alexa Fluor 488 at 1:1000 in blocking solution. Add DAPI to a final concentration of 10uM.
• 34.Incubate at 20°C–25°C for 45min, or 2hrs at 4°C.
• 35.Remove antibody solution and wash 3 times for 5min with PBS-T. Lastly, add 500uL of PBS-T.

Pause point: Cells can be stored at 4°C until imaging, but we recommend to image within 5 days for best image quality.

• 36.Acquire 2x2 10x stitched tile images in 405 and 488 channels in 3–5 random areas per sample (Figure 2).
• 37.
  To analyze images, open separate channels in ImageJ (Fiji) (Figure 2).

  • a.
    Produce binary images for each channel by Image>Adjust>Threshold. Adjust Threshold slider so that most of the signal is captured for both separate channels.
  • b.
    For the DAPI channel, separate individual nuclei by Process>Binary>Watershed.
  • c.
    To calculate the Differentiation Index, the amount of myotubes formed after differentiation: Analyze>Analyze Particles. To include all myotubes, set parameters to the following:
    Size: 0-Infinity.
    Circularity: 0.00–1.00.
    Select “Display results” and record total area.
  • d.
    To calculate the Fusion Index, the number of nuclei fused to form the myotube:

    • i.
      Obtain a binary image of nuclei present within myotubes by Process>Image Calculator>Select the DAPI channel, select “AND”, and select the MF-20 channel>OK
    • ii.
      This provides an image with non-specific small areas (Figure 3A, orange arrowheads) as well as incomplete and peripheral nuclei (Figure 3A, cyan arrowheads).
    • iii.
      To remove small debris, restrict the “size” component under Analyze>Analyze Particles to Size: 15-Infinity.
    • iv.
      To remove the remaining incomplete and peripheral nuclei (Figure 3B, cyan arrowheads), increase the “circularity” component under Analyze>Analyze Particles to Circularity: 0.6–1 or until peripheral nuclei are excluded (Figure 3C).

      Note: As experiments can slightly differ between biological replicates, we normalize results to their control and report percent (%) change between the groups.

      Proceed to troubleshooting 6 for more information.

Figure 2.

ImageJ analysis pipeline for Differentiation and Fusion indices

(A) Example of field of view of image acquired in step 36, and a zoomed in area. Immunofluorescence of mature myotubes (MF20, red) and nuclei (DAPI, blue). Scale bars: 250μm and 50μm, respectively.

(B) Analysis of images to obtain a Differentiation Index and Fusion Index. Green arrowheads show examples of myonuclei after image processing.

Figure 3.

Size and circularity exclusion used to quantify myonuclei in ImageJ

(A) Binary image generated using the Image Calculator, with particle outlines produced by Analyze Particles without any exclusion. Small debris (orange arrowheads) and peripheral/incomplete nuclei (cyan arrowheads).

(B) Particle outlines after applying size exclusion to remove small debris.

(C) Particle outlines after applying circularity exclusion to remove irregularly shaped or incomplete nuclei.

Expected outcomes

This protocol was developed to obtain primary FAPs following muscle injury and to co-culture them with the C2C12 cell line to validate co-culture experiments with the established fibroblast cell line 3T3-L1. The amount of FAPs obtained after differential plating depends the most on the degree of injury and how many muscles (TA and/or Gastrocnemius) are used per sample. If using 4 injured TAs or 1 TA and Gastrocnemius cultured in a P100/T75, expect 80% confluency in 2–3 days.

Limitations

This protocol is optimized for isolating FAPs from young adult 129S1/SvlmJ mice (9-13 weeks old) following a CTX or GLY injury. Digestion efficiency and yield may vary with age, strain, or mouse model, especially if extracellular matrix composition differs. Extent of injury heavily influences yield of isolated FAPs and should be taken in consideration when harvesting before isolating FAPs. We have previously shown that CTX and GLY differ in signaling pathways and phenotypic outcomes^26,27,28; we have not compared how isolated FAPs from either injury affect myogenesis in vitro. FAPs can also be isolated from uninjured muscle but digestion and amount of muscle must be optimized. This approach provides a mixed population of FAPs and does not resolve subpopulation specific effects on myogenesis. Lastly, the analysis pipeline described is not exact in the detection of myonuclei, leading to under- or overestimation depending on image handling and advise to treat all images equally. Accuracy can be improved by analyzing multiple, large fields of view to provide a reliable representation of overall differences between groups. Incorporation of emerging AI-based tools may further enhance accuracy and reduce the number of images required.

Troubleshooting

Problem 1

Insufficient injury to muscles (Step 10).

Potential solution

Before injuring to isolate primary FAPs, practice the injury on culled mice with a 1:1 solution of diluted black ink (1:5 dilution in PBS) and Glycerol (end concentration 50%). Once injuries are consistent, proceed to injure mice to harvest FAPs.

Problem 2

Too much debris and clogging of filter (Step 12).

Potential solution

First filter the suspension through a 100 μm filter before proceeding with a 70 μm filter.

Problem 3

Few cells obtained after allowing them to adhere (Step 20).

Potential solution

This could be due to multiple factors; little muscle injury will result in few cells, as well as over- or under digestion times. Too much mechanical digestion can lead to higher levels of cellular damage, while excessive enzymatic digestion can be harsh on cells. Under digestion occurs if large pieces of tissue remain and the digestion buffer remains clear/transparent.

Problem 4

FAPs rapidly differentiate after plating (Step 22).

Potential solution

Once FAPs have adhered after initial isolation, supplement fibroblast cell media with rFGF at 5ng/mL upon isolation to promote a pro-proliferative and undifferentiated state.

Problem 5

Contamination after FAP isolation (Step 22).

Potential solution

Contamination can most likely be attributed during initial muscle harvest. Ensure thorough application of 70% ethanol on the lower half of the body of the mouse. Additionally, shaving the whole lower half of the body in a separate area before thorough ethanol application can aid in minimizing contamination from hair.

Problem 6

Accurately capturing myonuclei during analysis (step 37).

Potential solution

The parameters to quantify myotube nuclei for Fusion Index may need to be slightly optimized to capture nuclei within the myotubes, while excluding partial or peripheral nuclei. Before proceeding to analyze all images from an experiment, optimize parameters in an initial first image. Adjust the minimum size in Size Parameter to avoid detection of small DAPI positive areas or partial nuclei; and adjust the Circularity Parameter to exclude partial nuclei that overlap with MF20 staining but are not within the myotube.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Daniel Kopinke (dkopinke@ufl.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Alessandra M. Norris (alessandra.norris.1@vanderbilt.edu).

Materials availability

This study did not generate new or unique reagents.

Data and code availability

This protocol did not generate data or code.

Acknowledgments

We thank the members of the Kopinke laboratory for critical reading of the manuscript. This work was supported by the US National Institutes of Health (NIH) grants 1R01AR079449 to D.K. and T32HD043730 to A.M.N. D.K. was also supported by the UF Thomas Maren Junior Research Excellence Fund. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The graphical abstract and Figure 1 were created with BioRender.

Author contributions

Conceptualization, A.M.N. and D.K.; investigation, A.M.N. and A.B.A.; writing – original draft, A.M.N.; writing – review and editing, A.M.N. and D.K.; funding acquisition, D.K.

Declaration of interests

The authors declare no competing interests.