Immunofluorescence analysis of myogenic differentiation

Abstract

Skeletal muscle is a highly regenerative tissue that can efficiently recover from various damages caused by injuries and excessive exercises. In adult muscle, stem cells termed satellite cells are mitotically quiescent but activated upon muscle damages to enter the cell cycle as myogenic precursor cells or myoblasts. After several rounds of cell cycles, they exist the cycle and fuse to each other to form multinucleated myotubes, and eventually mature to become contractile myofibers. Satellite cells can be readily isolated from mouse skeletal muscle with enzymatic digestion and magnetic separation with antibodies against specific surface markers. C2C12 cells are an immortalized mouse myoblast cell line that is commercially available and more readily expandable than primary myoblasts. Both primary myoblasts and C2C12 cells have been extensively used as useful in vitro models for myogenic differentiation. Proper examination of this process requires monitoring specific protein expression in subcellular compartments, which can be accomplished through immunofluorescence staining. This chapter describes the workflow for the isolation of satellite cells from mouse skeletal muscle and subsequent immunofluorescence staining to assess the proliferation and differentiation of primary myoblasts and C2C12 cells.

1. Introduction

Skeletal muscle is the most abundant tissue in the body and possesses a tremendous regenerative capacity. It is composed of several types of cells, including vascular cells, fibroblasts, and immune cells, in addition to multinucleated myofibers, the main structural and functional component, and the mononuclear muscle stem cells called satellite cells [1–3]. In particular, satellite cells play the central role during muscle repair, regeneration, and remodeling triggered by injuries and diseases. These cells exist in adult skeletal muscle as mitotically quiescent cells between the plasma membrane of myofibers and the basal lamina (basement membrane). Upon activation by muscle damages, satellite cells re-enter the cell cycle as myogenic precursor cells or myoblasts. After expanding the pool size, myoblasts re-exit the cell cycle, start expressing muscle functional proteins, and fuse to form new myotubes or expand existing myofibers, restoring the lost muscle volume. The myotubes continuously enlarge through additional fusions and increase cytoplasmic volume per nucleus during maturation, forming contractile myofibers. In addition, some myoblasts revert to satellite cells as a stem cell pool, thereby maintaining the regenerative capacity of skeletal muscle throughout life (self-renewal capacity).

Isolated satellite cells can be readily cultured in vitro, expanded, and passaged at least 20 times. They can also undergo myogenic differentiation, fusion, and self-renewal, recapitulating the in vivo behaviors [4, 5]. The widely used methods of purifying satellite cells require time-consuming and relatively harsh fluorescence-activated cell sorting (FACS) [6, 7]. Therefore, we describe here a more rapid and less harsh protocol using magnet-activated cell sorting (MACS) with antibodies against cell surface markers as an alternative [8].

C2C12 cells are an immortalized myoblast cell line originated from adult mouse satellite cells that has been frequently used as a commercially available alternative to primary myoblasts. Both primary myoblasts and C2C12 cells have been widely used to address a variety of questions in muscle biology in vitro, including differentiation [9–12], regeneration [4, 13], muscle growth [14], maturation [15], stem cell self-renewal [16, 17], muscular dystrophies [15], muscle aging [3, 18], formation of neuro-muscular junctions [19], and transdifferentiation into adipocytes or osteocytes [13].

In this chapter, we describe protocols for MACS-based preparation of highly purified satellite cells from adult mouse skeletal muscle, differentiation of primary myoblasts and C2C12 cells, and immunofluorescence staining of the cells after differentiation.

2. Materials

2.1. Reagents for cell culture

1. C2C12 cells, American Type Culture Collection, CRL-1772

2. C57BL/6J strain mouse, 8–10 weeks-old male or female, Jackson Laboratory, 000664

3. Phosphate-buffered saline without calcium and magnesium (PBS). Corning, 21-040-CV

4. 10X PBS, Thermo Fisher Scientific, 70011044

5. Dulbecco’s Modified Eagle’s Medium (DMEM), Corning, 10-013-CV

6. HAM’s F-10 medium, Thermo Fisher Scientific, 11550043

7. HyClone defined fetal bovine serum (FBS), Cytiva, SH30070.03

8. 0.25% trypsin-EDTA, Thermo Fisher Scientific, 25200056

9. Horse serum (HS), Thermo Fisher Scientific, 16050122, heat-inactivated by incubation in a water bath at 56 °C for 30 min. (see Note 1)

10. 500 μg/ml bFGF, Human FGF-basic (aa 1-155) recombinant protein, Thermo Fisher Scientific, PHG0263, dissolved in double-distilled water. Store in aliquots at −80 °C.

11. 10,000 U/ml Penicillin and 10,000 μg/ml Streptomycin, Thermo Fisher Scientific, 15140148

12. Myoblast growth medium. HAM’s F-10 medium with 20% FBS, 20 ng/ml bFGF, 100 U/ml penicillin, and 100 μg/ml streptomycin.

13. Myogenic differentiation medium. DMEM with 5% HS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

14. 0.01% Collagen solution. Dissolve rat tail collagen (BD Biosciences, 354236) in 0.2% acetic acid in double distilled water.

15. 0.2% Collagenase solution. Dissolve collagenase type 2 (Worthington, CLS-2) in DMEM with 10% FBS.

16. 70 μm Cell strainer. Thermo Fisher Scientific, 08-771-2

2.2. Reagents for the purification of satellite cells

• LD column, Miltenyi Biotec, 130-042-901

• MS column, Miltenyi Biotec, 130-042-201

• Anti-PE MicroBeads, Miltenyi Biotec, 130-048-801

• Anti-biotin MicroBeads, Miltenyi Biotec, 130-090-485

• MACS MultiStand, Miltenyi Biotec, 130-042-303

• MACS buffer containing 2 mM EDTA and 0.5% bovine serum albumin (BSA) in PBS

2.3. Reagents for immunofluorescence staining

1. 4% paraformaldehyde. Add 2 g of paraformaldehyde and 20 μl of 5N sodium hydroxide to 45 ml double distilled water. Dissolve by heating in a microwave oven for 2 min. Add 5 ml 10X PBS.

2. Permeabilization solution. 0.5 % Triton X-100 in PBS.

3. Blocking solution. 1% BSA in PBS

4. Washing solution. 0.01% Triton X-100 in PBS.

5. 1 μg/ml 4’,6’-diamidine-2’-phenylindole dihydrochloride (DAPI) dissolved in PBS, MilliporeSigma, 10236276001

6. Click-iT EdU Alexa Fluor 488 Imaging Kit, Thermo Fisher Scientific, C10337

7. Dako Flurorescent Mounting Medium, Agilent Technologies, S302580-2

2.4. Antibodies

See Table 1.

Table 1.

Antibodies

Protein	Manufacturer	Catalog #	Dilution (fold)
CD31-PE	eBioscience	12-0311	200
CD45-PE	eBioscience	12-0451	200
Sca1-PE	eBioscience	12-5981	200
Integrin α7-biotin	Miltenyi Biotec	130-102-125	200
MyHC	Developmental Studies Hybridoma Bank	MF 20	50
MyoD	Santa Cruz Biotechnology	sc-304	500
Alexa Fluor 488 donkey anti-mouse IgG	Thermo Fisher Scientific	A-21202	1000
Alexa Fluor 594 donkey anti-rabbit IgG	Thermo Fisher Scientific	A-21207	1000

3. Methods

3.1. Preparation and culture of primary myoblasts

Satellite cells are purified from mouse muscle as CD31(−), CD45(−), Sca-1(−), and Integrin 7α(+) cells with the following protocol.

1. Mince the whole hind limb muscle of 8–10 weeks old mice into a smooth pulp using a pair of ophthalmology scissors. Approximately 5 × 10⁶ dissociated cells can be obtained from one mouse. (see Note 2)

2. Incubate it in 5 ml of 0.2% collagenase at 37 °C for 60 min to dissociate the cells.

3. Triturate the cell clusters via an 18 G needle and incubate the cell suspension at 37 °C for 15 min.

4. Triturate the cell suspension via an 18 G needle again to dissociate it into a single-cell suspension. Add 40 ml of 2% FBS in DMEM to quench the collagenase.

5. Transfer the cell suspension onto a 70 μm cell strainer on a 50 ml tube and pipette the cell suspension up and down on the filter until it passes through the strainer.

6. Centrifuge the tube at 670 ×g (2,000 rpm) at 4 °C for 5 min. Aspirate and discard the supernatant.

7. Resuspend the cells with 10 ml of 2% FBS in DMEM and centrifuge the tube again. Aspirate and discard the supernatant.

8. Resuspend the cells with 200 μl of 2% FBS in DMEM and transfer the cell suspension into a 1.5 ml microcentrifuge tube.

9. Add 1 μl each of CD31-PE, CD45-PE, Sca-1-PE, and Integrin α7-biotin antibodies and place the tube on ice for 30 min. See Table 1 for the dilution of the antibodies.

10. Add 1 ml of 2% FBS in DMEM and centrifuge at 360 ×g (2,000 rpm) at 4 °C for 3 min. Aspirate and discard the supernatant. Repeat this step once.

11. Resuspend the cells with 200 μl of 2% FBS in DMEM and add 10 μl of Anti-PE MicroBeads. Incubate on ice for 30 min.

12. Add 1 ml of MACS buffer and centrifuge at 360 ×g at 4 °C for 3 min. Aspirate and discard the supernatant. Repeat this step once and resuspend the cells in 1ml of MACS buffer.

13. Set up an LD column on a MACS MultiStand and rinse the column with 2 ml of MACS buffer.

14. Transfer the cell suspension onto the LD column and collect the flow-through into a 1.5 ml tube. The flow-through contains CD31(−), CD45(−), and Sca-1(−) cells, including satellite cells.

15. Centrifuge the flow-through at 360 ×g at 4 °C for 3 min and discard the supernatant.

16. Resuspend the cells with 200 μl of 2% FBS in DMEM, add 10 μl of Anti-biotin MicroBeads, and incubate on ice for 30 min.

17. Add 1 ml of MACS buffer and centrifuge at 360 ×g at 4 °C for 3 min. Discard the supernatant and repeat this step. Resuspend the cells with 500 μl of MACS buffer.

18. Set up an MS column on a MACS MultiStand and rinse the column with 500 μl of MACS buffer.

19. Transfer the cell suspension onto the MS column and discard the flow-through containing Integrin α7(−) cells.

20. Rinse the column with 1 ml of MACS buffer and repeat this step.

21. Remove the column from the magnetic field of the MACS MultiStand. Load 1 ml of MACS buffer onto the column and elute Integrin α7(+) cells into a 1.5 ml microcentrifuge tube by pushing the syringe plunger from the top of the column. Repeat the elution with 1.5 ml of MACS buffer.

22. Centrifuge the combined eluates at 360 ×g at 4 °C for 3 min and discard the supernatant.

23. Isolated satellite cells are cultured as primary myoblasts in collagen-coated culture dishes in Myoblast growth medium at 37 °C with 5% CO₂. For collagen coating, add 0.01% collagen into culture dishes and removed it after incubation for 3 hr at room temperature. Dry the collagen-coated dishes completely before use. (see Note 3)

24. Change the medium every other day. Four days after seeding, harvest the cells with 0.25% trypsin and re-seed into collagen-coated dishes at 0.5–1×10⁶ cells/10 cm dish to maintain the culture. Freeze the rest of the cells in 90% FBS and 10% DMSO and store in a liquid nitrogen tank.

3.2. Differentiation of primary myoblasts

1. Use low-passage primary myoblasts (typically less than eight passages) for differentiation and immunostaining.

2. Day -1: Seed 1×10⁵ cells/well in collagen-coated 6-well plates in Myoblast growth medium.

3. Day 0: The cells are approximately 20% confluent. Wash the cells with PBS twice and add Myogenic differentiation medium. Replace the medium every other day.

4. Day 1, 3, and 5: Remove the culture medium and wash the cells with PBS once. Fix the cells with 4% paraformaldehyde for 10 min. Rise the cells with PBS twice and store the plates in plastic wrap at 4 °C until use for immunofluorescence staining.

3.3. Differentiation of C2C12 cells

1. Maintain C2C12 cells with 10% FBS in DMEM in a 37 °C and 5% CO₂ incubator.

2. Day -1: Seed 1×10⁵ cells/well in 12-well plates.

3. Day 0: The cells are 90% confluent. (see Note 4). Wash the cells with PBS twice and add Myogenic differentiation medium. Replace the medium every other day.

4. Day 1, 3, and 5: Remove the culture medium and wash the cells with PBS once. Fix the cells with 4% paraformaldehyde for 10 min. Rise the cells with PBS twice and store the plates in plastic wrap at 4°C until use for immunofluorescence staining.

4. Immunofluorescence staining of the cells

4.1. Immunofluorescence staining of the cells

• 1.Treat the cells with the permeabilization solution for 10 min.
• 2.Treat the cells with the blocking solution for 30 min.
• 3.Stain the cells with antibodies against anti-sarcomeric myosin heavy chain (MyHC) and MyoD diluted in the washing solution at 4°C overnight. See Table 1 for the dilution of the antibodies.
• 4.Wash the cells with the washing solution twice.
• 5.Incubate the cells with Alexa Fluor 488 anti-mouse IgG and Alexa Fluor 594 anti-rabbit IgG diluted in the washing solution at room temperature for 1 hr.
• 4.Wash the cells with the washing solution twice.
• 5.Counterstain DNA with 1 μg/ml DAPI for 30 min.
• 6.Store the cells in PBS or add Fluorescent Mounting Medium and place a cover glass on the cells.
• 7.To quantify the proliferation of myoblasts, pulse-label the cells with 1 μg/ml of EdU for 3 hrs before fixation. Detect EdU with a Click-iT EdU Alexa Fluor 488 Imaging Kit. Apply immunofluorescence staining as described above to identify myoblasts after the detection of EdU. Use Alexa Fluor 555 anti-mouse IgG, instead of Alexa Fluor 488 anti-mouse IgG, to detect MyHC.

4.2. Quantification of the differentiation

Differentiation index is defined as a percentage of nuclei existing within MyHC(+) cells. Fusion index is a percentage of nuclei located in MyHC(+) cells containing more than one nuclei. Differentiation index and fusion index are analyzed with biological triplicates, including 500 – 1,000 nuclei in each replicate.

5. Notes

1. Heat inactivation of HS promotes myogenic differentiation.

2. Skeletal muscle contains connective tissues including muscle membrane and tendons. Careful removal of the connective tissues before mincing facilitates the enzymatic dissociation and eventually increases the yield of purified satellite cells.

3. Following the MACS-based isolation, yield of satellite cells is approximately 1–2×10⁵ cells/mouse. There will be some blood cell contamination, which will disappear after the first passage of the cells.

4. C2C12 cells do not efficiently differentiate at a lower cell density unlike primary myoblasts. In addition, a differentiation-resistant population will be spontaneously selected during long-term culture of C2C12 cells. It is important to freeze multiple aliquots and thaw a new aliquot after a defined period of culture to obtain reproducible results.

Figure 1. Immunofluorescence staining of undifferentiated and differentiating primary myoblasts.

(A) Primary myoblasts cultured in Myoblast growth medium contain an EdU(+) proliferating population and a small number of spontaneously differentiating MyHC(+) cells.

(B) Primary myoblasts cultured in Myogenic differentiation medium for 1 and 3 days exhibit MyoD(+) and MyHC(+) terminally differentiating myocytes and multinucleated myotubes. DNA was counterstained with DAPI. Scale bar, 50 μm.