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Aug 2019

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Isolation, Culture, and Differentiation of Primary Myoblasts Derived from Muscle Satellite Cells
肌卫星细胞原代成肌细胞的分离、培养和分化   

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Abstract

The skeletal muscle is key for body mobility and motor performance, but aging and diseases often lead to progressive loss of muscle mass due to wasting or degeneration of muscle cells. Muscle satellite cells (MuSCs) represent a population of tissue stem cells residing in the skeletal muscles and are responsible for homeostatic maintenance and regeneration of skeletal muscles. Growth, injury, and degenerative signals activate MuSCs, which then proliferate (proliferating MuSCs are called myoblasts), differentiate and fuse with existing multinuclear muscle cells (myofibers) to mediate muscle growth and repair. Here, we describe a protocol for isolating MuSCs from skeletal muscles of mice for in vitro analysis. In addition, we provide a detailed protocol on how to culture and differentiate primary myoblasts into myotubes and an immunofluorescent staining procedure to characterize the cells. These methods are essential for modeling regenerative myogenesis in vitro to understand the dynamics, function and molecular regulation of MuSCs.

Keywords: Satellite Cell (卫星细胞), Myoblast (成肌细胞), Self-renewal (自我更新), Differentiation (变异), Skeletal Muscle (骨骼肌), Pax7 (Pax7抗体), MyoG (MyoG抗体)

Background

Muscle homeostasis, maintained by diverse cellular functions, is vital for preserving muscle integrity. Tissue specific adult stem cells are capable of regenerating local tissues continuously throughout life. In adult skeletal muscles, a population of stem cells called muscle satellite cells (MuSCs) possess a robust regenerative capacity that is key for muscle homeostasis (Yin et al., 2013; Dumont et al., 2016). Quiescent MuSCs reside in a niche beneath the basal lamina juxtaposed to the muscle fiber and are responsible for growth and regeneration of muscle (Yin et al., 2013; Dumont et al., 2016).

MuSCs are a heterogeneous population with diverse cellular and molecular profiles. Quiescent MuSCs, identified by the expression of Pax7, are activated and enter a proliferating stage in response to external stimuli such as growth factors, injury, or pathological conditions (Kuang et al., 2008). The proliferating myoblasts undergo several rounds of cell division and terminally differentiate into mononuclear myocytes, which fuse into myotubes to mediate muscle repair (Rudnicki et al., 2008). A dynamic balance between asymmetric and symmetric division is also important for determining the fate of myogenic cells in terms of self-renewal and expansion (Kuang et al., 2007).

Primary myoblasts (referring to MuSCs that are isolated from muscle) and the derived myotubes are important for modeling MuSC function and muscle physiology in vitro. Several methods for the isolation of primary myoblasts have been reported to investigate their cell biology (i.e., proliferation, self-renewal and differentiation) and molecular regulation. One method involves enzymatic digestion of muscle tissues to liberate single myofibers that carry MuSCs to model in vivo behavior of MuSCs while they are still in their native niche (Shefer et al., 2005). Another method involves fluorescence-activated cell sorting (FACS), an efficient and widely used technique for selecting rare cell populations from various tissues. FACS-mediated purification of MuSCs from other muscle resident cells such as fibroblasts and immune cells involves the use of positive selection markers (e.g., CD34, Integrin α7 and Vcam1) and negative selection markers (e.g., CD45, CD31, CD11b, and Sca1) (Liu et al., 2015). FACS requires a flow cytometer machine that is not available to many labs, and a prolonged sorting process that often reduces the viability of cells. Alternatively, MuSCs and primary myoblasts can be enriched using other more affordable methods, such as magnetic activated cell sorting (MACS) (Motohashi et al., 2014). However, MACS also requires antibodies and beads to eliminate non-muscle cells, and the number of cells that can be isolated is limited by the column and the speed of MACS isolation is slow as the mixture of cells flow through MS column by gravity. Finally, primary myoblasts can be enriched simply through a procedure of serial pre-plating based on differential affinity of different cell types to culture substrate.

Here, we describe a protocol for convenient and efficient purification of primary myoblasts from hind limb muscles of mice (Yue et al., 2017; Jia et al., 2019). This method consists of mechanical mincing followed by enzymatic digestion to release mononuclear cells, and selection of MuSCs by FACS. The process of primary myoblast isolation includes muscle dissection and digestion by collagenase type II and dispase, followed by myoblast purification through multiple rounds of pre-plating. Pre-plating is essential to purify a population of MuSCs on the basis of adhesion characteristics. We will also describe how to culture, differentiate, and validate pre-differentiation myoblasts and differentiated multinucleated myotubes using immunofluorescent staining for the expression of myogenic regulatory markers.

Materials and Reagents

  1. Dissection boards (Styrofoam board)
  2. 6-well tissue culture (TC) plates (Falcon, catalog number: 353046 )
  3. Mounting medium (Diagnostic BioSystems, catalog number: K024 )
  4. 11 mm x 11 mm cover slides (IMEB, catalog number: CG1-2450 )
  5. 60 mm TC plates (Falcon, catalog number: 353002 )
  6. 100 mm TC plates (Falcon, catalog number: 353003 )
  7. 1.5 ml Eppendorf tubes (DOT Scientific, catalog number: RN1700-GMT )
  8. 15 ml polystyrene centrifuge tubes (Falcon, catalog number: 352095 )
  9. 50 ml high Clarity PP centrifuge tubes (Falcon, catalog number: 352098 )
  10. 70 µm cell strainers (Falcon, catalog number: 352350 )
  11. 10 ml serological pipettes (Falcon, catalog number: 357551 )
  12. Mouse aged 4-6 weeks
  13. Phosphate-buffered saline (PBS, pH 7.4) (Sigma-Aldrich, catalog number: P3813 )
  14. Tween-20 (VWR, catalog number: 97062332 )
  15. Dispase II (Roche Applied Science, catalog number: 0 4942078001 )
  16. Collagenase type II (Worthington, catalog number: LS004177 )
  17. 0.25% Trypsin (Gibco, catalog number: 25200056 )
  18. Fetal bovine serum (FBS) (Hyclone, catalog number: SH3007103 )
  19. Horse serum (HS) (Hyclone, catalog number: SH3007403 )
  20. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, D5796 )
  21. Ham's F-10 Nutrient Mixture (Gibco, catalog number: 11550043 )
  22. Collagen type I (Sigma-Aldrich, catalog number: C7661 )
  23. Matrigel (Corning, catalog number: 354234 )
  24. Paraformaldehyde powder (Polysciences, catalog number: 00 380 )
  25. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, catalog number: D8417 )
  26. Penicillin-streptomycin solution (Hyclone, catalog number: SV30010 )
  27. Glycine (Sigma-Aldrich, catalog number: G8898 )
  28. Ethanol (Decon Labs, catalog number: V1001 )
  29. Goat serum (Jackson ImmunoResearch, catalog number: 005-000-121 )
  30. Bovine serum albumin (BSA) (Gemini Bio-Products, catalog number: 700-105P )
  31. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  32. Sodium azide (Fisher Scientific, catalog number: S2271 )
  33. Basic fibroblast growth factor (bFGF) (Promega, catalog number: G5071 )
  34. Primary antibody:
    Pax7 (Developmental Studies Hybridoma Bank)
    MyoD (Santa Cruz Biotechnology, catalog number: sc-760 )
    MyoG (F5D) (Developmental Studies Hybridoma Bank)
    MyHC (MF20) (Developmental Studies Hybridoma Bank)
  35. Secondary antibody:
    Goat anti-Mouse IgG1, Alexa Fluor 568 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21124 )
    Goat anti-Mouse IgG2b, Alexa Fluor 488 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21141 )
    Goat anti-Rabbit IgG, Alexa Fluor 488 (Thermo Fisher Scientific, Invitrogen, catalog number: A-11034 )
  36. 70% ethanol (see Recipes)
  37. Collagen type I coated TC plates (see Recipes)
  38. Matrigel coated TC plates (see Recipes)
  39. Digestion Medium (see Recipes)
  40. Neutralization medium (see Recipes)
  41. Growth medium (see Recipes)
  42. Differentiation medium (see Recipes)
  43. 0.05% Trypsin (see Recipes)
  44. 4% Paraformaldehyde (PFA) (see Recipes)
  45. 100 mM glycine solution (see Recipes)
  46. Blocking buffer (see Recipes)
  47. bFGF stock (see Recipes)
  48. PBS-T (see Recipes)

Equipment

  1. Dissecting scissors, 10-cm, straight (WPI, catalog number: 14393-G )
  2. Tweezers, 4.75”, straight (Excelta, catalog number: 3-S-SE )
  3. T-pins (Business source, catalog number: 32351 )
  4. Swing-bucket centrifuge (Thermo Scientific, model: CL2 )
  5. Autoclave (Beta Star life sciences, model: small sterilizer series 26” x 26” model )
  6. Class II, type A2 biosafety cabinet (Labconco, catalog number: 3440009 )
  7. Thermostat water bath (Thermo Fisher Scientific, catalog number: TSGP02 )
  8. Cell incubator (Thermo Fisher Scientific, model: Heracell 240 )
  9. 14.0 MP digital USB microscope camera (OMAX Microscope, catalog number: A35140U3 )
  10. Leica DMI 6000B fluorescent microscope (Leica Microsystems, model: DMI 6000 B )
  11. Lumen 200 fluorescence illumination system (Prior Scientific, model: Lumen 200 )
  12. Coolsnap HQ CCD camera (Photometrics, model: Coolsnap HQ )
  13. Ultra pipet controller (Corning, catalog number: 4099 )
  14. 10 μl, 200 μl, 1,000 μl pipettes (Gilson, model: Pipetman )

Software

  1. Photoshop software (e.g., Photoshop CC)

Procedure

  1. Preparation prior to isolation
    1. 70% ethanol (see Recipe 1).
    2. Autoclave 1.5 ml Eppendorf tubes.
    3. Sterilize tools with 70% ethanol.
    4. Collagen type I to coat cell culture plate (see Recipe 2).
    5. Matrigel to coat cell culture plate (see Recipe 3).
    6. Plate coating
      1. Coat 100 mm plates with 5 ml of collagen type I to cover the plate completely for 30 min at room temperature. After 30 min, transfer collagen type I to the original tube, and air-dry the plate. The plate can be stored in A2 biosafety cabinet until ready to use. This plate will be used for pre-plating.
        Note: Collagen type I can be reused for future use.
      2. Coat 60 mm plates with 3 ml of Matrigel to cover the plate completely for 20 min at room temperature. After 20 min, transfer Matrigel to the original tube, and air-dry the plate. It is recommended to use the plate right after Matrigel is dried out. This plate will be used for differentiation of myoblasts.
        Note: Matrigel can be reused for future use.
    7. Digestion medium (see Recipe 4).
    8. Neutralization medium (see Recipe 5).
    9. Growth medium (see Recipe 6).
    10. Differentiation medium (see Recipe 7).
    11. 0.05% trypsin (see Recipe 8).
    12. 4% paraformaldehyde (PFA) fixation solution (see Recipe 9).
    13. 100 mM glycine solution (see Recipe 10).
    14. Blocking buffer (see Recipe 11).
    15. bFGF (see Recipe 12).
    16. PBS-T (see Recipe 13).

  2. Muscle dissection
    1. Sacrifice a mouse aged 4-6 weeks following the approved protocol in your laboratory. In this case, cervical dislocation is used.
      Note: Dissection of a mouse does not need to be performed inside an A2 biosafety cabinet.
    2. Spray the mouse with 70% ethanol and pin the mouse face up on a dissection board.
    3. Cut the skin at the ankle using sterilized scissors. With the mouse in a transverse abdominal position, use tweezers to expose the quadricep muscle. Collect muscles in cold PBS. Flip the mouse and pin it with its back facing up. Remove the skin on lower body, dissect two entire hind limb skeletal muscles and place them in cold PBS.
    4. Transfer the dissection tools and the plate containing muscle to a biosafety cabinet. Wipe all dissection tools with 70% ethanol to avoid potential contamination from outside of the biosafety cabinet.

      Video 1. Muscle dissection of mouse. This video demonstrates a detailed procedure of how to isolate hind limb muscles, as well as how to remove the epimysium and fat around the muscle and the neural bundles within the muscle. After transferring the muscle to a 60 mm plate containing PBS, it was washed several times in PBS, minced with a pair of scissors or a razor blade, and digested by collagenase type II to liberate MuSCs from myofibers.

  3. Muscle digestion
    1. Wash hind limb muscles with PBS until muscles are free of hair.
      Note: It is recommended to wash several times until hair is completely removed from muscle tissue.
    2. Blot dry muscles on tissue paper.
    3. Transfer muscle to 1.5 ml Eppendorf tube.
    4. Mince the muscle using dissection scissors inside a 1.5 ml Eppendorf tube.
      Note: Finely minced muscle will yield more myoblasts.
    5. Transfer the minced muscle into a 15 ml Falcon tube.
    6. Digest the muscle with digestion medium (5 ml per mouse) in a 37 °C water bath for 12 min, shaking the tube every 2 min.
    7. Mix the digested muscle with a 10 ml serological pipette until the mixture can be smoothly pipetted.
    8. Digest for another 12 min in the 37 °C water bath, shaking the tube every 2 min.
    9. Stop digestion by adding 5 ml neutralization medium.
    10. Place a 70 µm sterilized cell strainer on top of a 50 ml tube, and pre-wet the strainer with 3 ml neutralization medium.
      Note: Pre-wetting is important for cells to pass smoothly through the cell strainer.
    11. Collect the media containing cell mixture using a 10 ml serological pipette and filter through the 70 µm sterilized cell strainer.
      Note: Collect cell filtrate underneath cell strainer using 1 ml pipette.
    12. Spin the cell mixture at 2,000 x g for 5 min at room temperature.
    13. Discard the supernatant using a vacuum pipette.
      Note: Do not aspirate the cell pellet.
    14. Resuspend the cell pellet with 5 ml growth medium.
    15. Seed the cells from one mouse in a non-coated 100 mm culture plate and incubate at 37 °C for 4 days, and supplement 5 ml of growth medium on top of pre-existing medium each day for 3 days.
      Note: A heterogeneous population of cell mixture and broken fibers will be visualized under a microscope on Day 4.

  4. Primary myoblast isolation and pre-plating
    1. On Day 4, small fibers pieces with rounded primary myoblasts can be seen, and cells are now ready for pre-plating.
    2. Mechanically detach cells attached to the bottom of plate by pipetting using a 10 ml serological pipette and transfer to a 50 ml tube. Floating cells are also transferred to the 50 ml tube.
      Notes:
      1. This method only utilizes mechanical detachment, therefore trypsin is not necessary.
      2. Confirm if most cells were detached from the plate by checking under a microscope.
    3. Centrifuge the 50 ml tube at 2,000 x g for 5 min.
      Note: Let centrifuge stop without applying brake. This usually takes a couple of minutes.
    4. Aspirate the media from the 50 ml tube.
    5. Add 3 ml trypsin into the 50 ml tube and resuspend cells carefully and incubate in a 37 °C water bath for 5 min.
      Note: Cells are still heterogeneous population.
    6. Centrifuge the 50 ml tube at 2,000 x g for 5 min.
    7. Discard the supernatant without disturbing the pellet and resuspend the cells with 5 ml of growth medium
    8. Transfer to a non-coated culture plate and incubate at 37 °C incubator for 45 min.
    9. After 45 min, transfer supernatant from the non-coated culture plate to collagen-coated plate.
      Note: Myoblasts do not adhere to the culture plate in a short time, therefore, transferring supernatant media helps to purify myoblast from other cells such as fibroblasts in the bottom of the plate.
    10. Steps D2-D9 (Pre-plating) can be repeated multiple times to obtain ≥ 95% purity of myoblasts.
      Note: Pre-plating can be repeated twice to increase myoblast purity. Myoblasts should appear small and rounded, while fibroblasts will appear elongated and at times have bipolar processes.

  5. Culturing and differentiation
    1. Culture the purified primary myoblasts in the growth medium, changing the media every 2 days. Maintain the cell density under 80% confluency to prevent the fusion of primary myoblasts.
      Note: Collagen type I coated plate is not required after pre-plating step.
    2. Prior to differentiation, coat a 60 mm culture plate with 3 ml of Matrigel for 20 min in the hood at room temperature.
    3. After removing Matrigel, allow for complete evaporation in the hood for 30 min.
      Note: It is not recommended to reuse Matrigel more than 5 times and should be stored at 4 °C or on ice when not in use.
    4. Incubate cells with 1 ml trypsin for 1 min at room temperature to detach primary myoblasts and seed cells onto Matrigel coated plate.
      Note: Primary myoblasts are cultured on Matrigel coated plate with 4 ml of growth medium until the desired cell density is reached.
    5. Once the confluency reaches 80-90%, change myoblast medium to differentiation medium.
    6. Maintain primary myoblasts with differentiation medium for the following three days.
      Note: Daily monitoring morphological change of primary myoblasts under a microscope is necessary. Clear morphological change from rounded primary myoblasts to elongated myotubes can be observed on Day 3.

  6. Immunostaining of Primary Myoblasts
    1. Place an autoclaved cover slide in a well of a 6-well plate.
    2. Seed myoblasts on the autoclaved cover slide
      Notes:
      1. Adherent myoblasts should be visible on the cover slide the following day.
      2. Myoblasts can be cultured several days to the desired confluence.
    3. Aspirate growth medium from the culture plate.
    4. Fix adherent cells by adding 1 ml of 4% PFA and incubate for 15 min at room temperature.
    5. Aspirate 4% PFA from the culture plate.
    6. Gently wash 3 times with 1 ml of PBS for 5 min each at room temperature.
    7. Aspirate PBS from the culture plate.
    8. Add 1 ml of 100 mM glycine and incubate for 10 min at room temperature.
    9. Remove 100 mM glycine and wash with 1 ml PBS for 5 min at room temperature.
    10. Repeat Step 9 for a total of 3 washes.
    11. Add 1 ml of blocking buffer and incubate for 1-2 h at room temperature.
    12. Add appropriate amount of primary antibody (Pax7-1:10, MyoD-1:300 in blocking buffer) and incubate overnight at 4 °C.
    13. Gently wash 3 times each with 1ml of PBS-T for 5 min at room temperature.
    14. Add appropriate amount of secondary antibody (Goat anti-mouse IgG1 for Pax7, MyoG, Goat anti-mouse IgG2b for MyoD, Goat anti-Rabbit IgG for MF20, DAPI-1:1,000 in PBS-T) at room temperature for 1 h.
      Note: Secondary antibodies are sensitive to light, so the steps below should be performed in a dark room.
    15. Gently wash 5 times each with 1 ml of PBS-T for 5 min at room temperature.
      Note: More washing is recommended to minimize background staining.
    16. Mount the coverslip with a drop of mounting medium and allow to dry.
    17. Evaluate staining under a fluorescent microscope.

Data analysis

This protocol includes visualization and evaluation of myoblasts in vitro before and after differentiation, after immunostaining with specific myogenic markers. Primary myoblasts cultured in growth medium were fixed by the addition of 4% PFA and were stained with Pax7 and MyoD, markers for undifferentiated myoblasts (Figure 1). After primary myoblasts were seeded on a Matrigel-coated plate, growth medium was changed to differentiation medium once the confluency of myoblasts reached 80%. After 3 days of differentiation, myoblasts were fixed and stained with MyoG and MF20, markers of early and late stages of myogenic differentiation, respectively (Figure 2). For research purposes, myoblasts can be stained with other antibodies, but it is recommended to co-stain with one of the myogenic markers to confirm the differentiation status of the myoblasts.


Figure 1. Phase contrast imaging and immunofluorescence staining of undifferentiated primary myoblasts. Representative phase contrast image (20x) demonstrates the morphology of undifferentiated primary myoblasts (A). Primary myoblasts were fixed for immunostaining after one day of cell culture. Representative immunofluorescence imaging using Pax7 (red) and MyoD (green) myogenic markers for undifferentiated primary myoblasts. Nuclei were stained with DAPI (blue) (B).


Figure 2. Phase contrast image and immunofluorescent staining of myotubes after differentiation for 3 days. A. Representative phase contrast image of differentiated myotube morphology (20x). B. Primary myoblasts were cultured on a Matrigel-coated plate for 3 days to form fully differentiated myotubes. For immunostaining, MyoG (red) and MF20 (green) were used as myogenic markers for differentiation to visualize multinucleated myotubes. Nuclei were stained with DAPI (blue).

Notes

  1. The yield of primary myoblasts isolated from young mice (3-4 weeks old) is higher than that of adult mice (2 months old)
  2. It is imperative that hair is completely removed during dissection and washing steps as excess hair can be a source of primary culture contamination.
  3. Finely cutting into small pieces of muscle tissues during the mincing step (Step C4) is helpful to increase myoblast yield.
  4. Additional rounds of pre-plating can be performed if myoblast purity is less than 95%.
  5. For staining, incubation of primary antibody at room temperature for 1 h is possible, but overnight incubation at 4 °C is strongly recommended.

Recipes

  1. 70% ethanol
    Add 70 ml of 100% ethanol in 30 ml ddH2O. Store at room temperature
  2. Collagen type I for coating
    1. Dissolve 0.1% collagen type I in 0.1 M acetic acid and stir the mixture at room temperature for 1 h to make a 10x stock solution
    2. 10x collagen type I stock solution is diluted to 1x in 0.1 M acetic acid to obtain 0.01% collagen type I
    3. Store at 4 °C
  3. Matrigel for coating
    Add 1.4 volumes DMEM medium to dilute Matrigel
    For 2.4 ml solution, dilute 1 ml Matrigel with 1.4 ml DMEM medium. Store at 4 °C
  4. Digestion Medium
    Prepare digestion medium by dissolving dispase II and collagenase type II in PBS at a concentration of 2.4 U/ml and 2.9 U/ml, respectively, and supplement with 2.5 mM CaCl2
    For 50 ml digestion medium, dissolve 125 mg dispase, 0.5 g collagenase type II, then add 125 μl 1 M CaCl2. Store at -20 °C
  5. Neutralization medium
    F-10 Ham’s medium is supplemented with 20% FBS, and 1% penicillin-streptomycin.
    For 50 ml neutralization medium, 39.5 ml F-10 Ham’s medium will be supplemented with 10 ml FBS and 500 μl penicillin-streptomycin. Store at 4 °C
  6. Growth medium
    F-10 Ham’s medium is supplemented with 20% FBS, 4 ng/ml basic fibroblast growth factor, and 1% penicillin-streptomycin
    For 50 ml growth medium, F-10 Ham’s medium will be supplemented with 10 ml FBS, 50 μl bFGF stock and 500 μl penicillin-streptomycin. Store at 4 °C
  7. Differentiation medium
    Dulbecco’s modified Eagle’s medium (DMEM) is supplemented with 2% horse serum, and 1% penicillin-streptomycin.
    For 50 ml differentiation medium, DMEM will be supplemented with 1 ml horse serum and 500 μl penicillin-streptomycin. Store at 4 °C
  8. 0.05% Trypsin
    For 50 ml 0.05% trypsin, add 10 ml of 0.25% trypsin in 40 ml autoclaved PBS
    Store at 4 °C
  9. 4% Paraformaldehyde (PFA)
    Dissolve 4% PFA (m/v) in PBS solution, stir the mixture at 60 °C water bath, and raise the pH to 7.4 by adding NaOH to make PFA powder more dissolvable
    For 100 ml 4% PFA, take 80 ml PBS solution, add 4 g PFA powder, dissolve the PFA as described above, and adjust the volume to 100 ml with PBS solution. Store at 4 °C
  10. 100 mM glycine solution
    Dissolve glycine in PBS at a concentration of 100 mM
    For 50 ml glycine solution, dissolve 3.75 g glycine in 50 ml PBS. Store at 4 °C
  11. Blocking buffer
    PBS supplemented with 5% goat serum, 2% BSA, 0.2% Triton X-100, 0.1% sodium azide
    For 100 ml blocking buffer, 5 ml goat serum, 2 g BSA, 2 ml 10% Triton X-100 and 0.1 g sodium azide is added in PBS. Aliquot and store at -20 °C
  12. bFGF stock
    Prepare bFGF stock by dissolving bFGF in serum-free medium at a concentration of 4 μg/ml (1,000x)
    For 4 μg/ml bFGF stock, 4 μg bFGF is dissolved in 1 ml of serum-free DMEM. Aliquot and store at -20 °C
  13. PBS-T
    Add 0.1% of Tween-20 in PBS
    For 1 L PBS-T solution, 1 ml Tween-20 is added in 1 L PBS. Store at room temperature

Acknowledgments

This protocol was adapted from our previous work published in Nature Communications (Yue et al., 2017) and Elife (Jia et al., 2019). This work was supported by grants from the National Institutes of Health of USA (R01AR071649) and the National Institute of Food and Agriculture of the United States Department of Agriculture (NC-1184).

Competing interests

The authors declare no competing interests.

Ethics

All procedures performed in animal study were guided by Purdue University’s Animal Care and Use Committee (PACUC) protocol number 1112000440.

References

  1. Dumont, N. A. and Rudnicki, M. A. (2016). Targeting muscle stem cell intrinsic defects to treat Duchenne muscular dystrophy. NPJ Regen Med 1.
  2. Jia, Z., Nie, Y., Yue, F., Kong, Y., Gu, L., Gavin, T. P., Liu, X. and Kuang, S. (2019). A requirement of Polo-like kinase 1 in murine embryonic myogenesis and adult muscle regeneration. Elife 8: e47097.
  3. Kuang, S., Kuroda, K., Le Grand, F. and Rudnicki, M. A. (2007). Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129(5): 999-1010.
  4. Kuang, S., Gillespie, M. A. and Rudnicki, M. A. (2008). Niche regulation of muscle satellite cell self-renewal and differentiation. Cell Stem Cell 2(1): 22-31.
  5. Liu, L., Cheung, T. H., Charville, G. W. and Rando, T. A. (2015). Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat Protoc 10(10): 1612-1624.
  6. Motohashi, N., Asakura, Y. and Asakura, A. (2014). Isolation, culture, and transplantation of muscle satellite cells. JOVE (86). DOI: 10.3791/50846.
  7. Rudnicki, M. A., Le Grand, F., McKinnell, I. and Kuang, S. (2008). The molecular regulation of muscle stem cell function. Cold Spring Harb Symp Quant Biol 73: 323-331.
  8. Shefer, G. and Yablonka-Reuveni, Z. (2005). Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol 290: 281-304.
  9. Yin, H., Price, F. and Rudnicki, M. A. (2013). Satellite cells and the muscle stem cell niche. Physiol Rev 93(1): 23-67.
  10. Yue, F., Bi, P., Wang, C., Shan, T., Nie, Y., Ratliff, T. L., Gavin, T. P. and Kuang, S. (2017). Pten is necessary for the quiescence and maintenance of adult muscle stem cells. Nat Commun 8: 14328.

简介

[摘要] 骨骼肌是身体活动和运动表现的关键,但是衰老和疾病通常会由于肌肉细胞的浪费或变性而导致肌肉质量的逐步丧失。肌卫星细胞(MuSCs)代表的组织STE群体米细胞小号居住在骨骼肌和负责骨骼肌的体内平衡维持和再生。生长,损伤和变性信号激活MuSC,然后增殖(增殖的MuSC被称为成肌细胞),分化并与现有的多核肌肉细胞(肌纤维)融合,以介导肌肉的生长和修复。在这里,我们描述了从小鼠骨骼肌中分离MuSC的体外实验方案分析。此外,我们提供了有关如何将原代成肌细胞培养和分化成肌管的详细协议,以及用于表征细胞的免疫荧光染色程序。这些方法对于在体外模拟再生肌生成以了解MuSC 的动力学,功能和分子调控至关重要。

[背景] 通过多种细胞功能维持肌肉的动态平衡,对于保持肌肉的完整性至关重要。组织特异性成体干细胞能够在整个生命中连续不断地再生局部组织。在成年骨骼肌中,称为肌肉卫星细胞(MuSC)的干细胞群具有强大的再生能力,这是肌肉动态平衡的关键(Yin 等人,2013; Dumont 等人,2016)。静态MuSC位于与肌肉纤维并列的基底层下方的壁iche中,负责肌肉的生长和再生(Yin 等人,2013; Dumont 等人,2016)。

MuSC是具有不同细胞和分子谱的异质群体。通过Pax7表达鉴定的静态MuSCs被激活并进入增殖阶段,以响应外部刺激,例如生长因子,损伤或病理状况(Kuang 等,2008)。增殖的成肌细胞经历数轮细胞分裂并最终分化为单核肌细胞,其融合入肌管以介导肌肉修复(Rudnicki 等,2008)。非对称分裂与对称分裂之间的动态平衡对于确定成肌细胞的自我更新和扩展的命运也很重要(Kuang et al 。,2007 )。

原代成肌细胞(指从肌肉中分离出来的MuSC )和衍生的肌管对于在体外模拟MuSC功能和肌肉生理非常重要。已经报道了几种分离原代成肌细胞的方法,以研究其细胞生物学(即增殖,自我更新和分化)和分子调控。一种方法涉及酶消化肌肉组织以释放携带MuSC的单肌纤维,以模拟当MuSC仍处于其天然位时的体内行为(Shefer 等人,20 05 )。另一种方法涉及荧光激活细胞分选(FACS),这是一种从各种组织中选择稀有细胞群体的有效且广泛使用的技术。FACS介导的从其他肌肉驻留细胞(例如成纤维细胞和免疫细胞)中纯化MuSC 涉及使用阳性选择标记(例如CD34,整合素α7 和Vcam1)和阴性选择标记(例如CD45,CD31,CD11b和Sca1) (Liu et al 。,2015)。FACS需要一个流式细胞仪机不可用的许多实验室,并且延长的排序处理,往往降低到细胞的活力。或者,可以使用其他更实惠的方法来富集MuSC和原代成肌细胞,例如磁激活细胞分选(MACS)(Motohashi 等人,2014)。但是,MACS还需要抗体和​​珠子来消除非肌肉细胞,并且可分离的细胞数量受到色谱柱的限制,并且由于细胞混合物通过重力流过MS色谱柱,因此MACS分离的速度很慢。最后,基于不同细胞类型对培养底物的不同亲和力,可以通过连续预铺的方法简单地富集原代成肌细胞。

在这里,我们描述了一种从小鼠后肢肌肉方便,有效地纯化原代成肌细胞的方案(Yue 等,2017; Jia 等,2019)。该方法包括的机械切碎,随后通过酶消化,以释放单核细胞,和MuSCs的选择被F ACS 。最初的成肌细胞分离过程包括肌肉解剖和II型胶原酶和分散酶消化,然后通过多轮预铺板纯化成肌细胞。根据附着特性,预镀对于纯化MuSC至关重要。我们还将描述如何使用免疫荧光染色培养,分化和验证分化前的成肌细胞和分化的多核肌管,以表达成肌调节标记。

关键字:卫星细胞, 成肌细胞, 自我更新, 变异, 骨骼肌, Pax7抗体, MyoG抗体

材料和试剂


 


1. 解剖板(发泡胶板)      


2. 6孔组织培养(TC)板(Falcon,目录号:353046)      


3. 固定介质(Diagnostic BioSystems,目录号:K024)      


4. 11毫米x 11毫米盖滑片(IMEB,目录号:CG1-2450)      


5. 60毫米TC板(猎鹰,目录号:353002)      


6. 100毫米TC 平板(Falcon,目录号:353003)      


7. 1.5毫升Eppendorf管(DOT Scientific,目录号:RN1700-GMT)      


8. 15毫升聚苯乙烯离心管(Falcon,目录号:352095)      


9. 50 ml高澄清度PP离心管(Falcon,目录号:352098)      


10. 70 µm细胞过滤器(Falcon,目录号:352350)   


11. 10毫升血清移液器(Falcon,目录号:357551)   


12. 4-6周龄的老鼠   


13. 磷酸盐缓冲盐水(PBS,pH 7.4)(Sigma-Aldrich,目录号:P3813)   


14. Tween-20(VWR,目录号:97062332)   


15. Dispase II(罗氏应用科学,目录号:04942078001)   


16. II型胶原酶(沃辛顿,目录号:LS004177)   


17. 0.25%胰蛋白酶(Gibco,目录号:25200056)   


18. 胎牛血清(FBS)(Hyclone,目录号:SH3007103)   


19. 马血清(HS)(Hyclone,目录号:SH3007403)   


20. Dulbecco改良的Eagle培养基(DMEM)(Sigma-Aldrich,D5796)   


21. Ham's F-10营养混合物(Gibco,目录号:11550043)   


22. I型胶原(Sigma-Aldrich,目录号:C7661)   


23. 基质胶(Corning,目录号:354234)   


24. 多聚甲醛粉末(Polysciences,目录号:00380)   


25. 4',6-二mid基-2-苯基吲哚二盐酸盐(DAPI)(Sigma-Aldrich,目录号:D8417)   


26. 青霉素-链霉素溶液(Hyclone,目录号:SV30010)   


27. 甘氨酸(Sigma-Aldrich,目录号:G8898)   


28. 乙醇(Decon Labs,目录号:V1001)   


29. 山羊血清(Jackson ImmunoResearch,目录号:005-000-121)   


30. 牛血清白蛋白(BSA)(Gemini Bio-Products,目录号:700-105P)   


31. Triton X-100(Sigma-Aldrich,目录号:X100)   


32. 叠氮化钠(Fisher Scientific,目录号:S2271)   


33. 碱性成纤维细胞生长因子(bFGF)(Promega,目录号:G5071)   


34.一抗:   


Pax7(发展研究杂交瘤银行)


MyoD(圣克鲁斯生物技术,目录号:sc-760)


MyoG(F5D)(发展研究杂交瘤库)


MyHC(MF20)(发育研究杂交瘤银行)


35. 二抗:   


              山羊抗小鼠IgG1,Alexa Fluor 568(Thermo Fisher Scientific,Invitrogen,目录号:A-21124)


山羊抗小鼠IgG2b,Alexa Fluor 488(Thermo Fisher Scientific,Invitrogen,目录号:A-21141)


山羊抗兔IgG,Alexa Fluor 488(Thermo Fisher Scientific,Invitrogen,目录号:A-11034)


36. 70%乙醇(请参阅食谱)   


37. I型胶原包被的TC板(请参阅食谱)   


38. 涂有Matrigel的TC板(请参阅食谱)   


39. 消化培养基(请参阅食谱)   


40. 中和介质(请参见食谱)   


41. 生长培养基(请参阅食谱)   


42. 鉴别介质(请参见食谱)   


43. 0.05%胰蛋白酶(请参阅食谱)   


44. 4%聚甲醛(PFA)(请参阅食谱)   


45. 100 mM甘氨酸溶液(请参阅食谱)   


46. 阻塞缓冲区(请参见食谱)   


47. bFGF库存(请参阅食谱)   


48. PBS-T(请参阅食谱)   


 


设备


 


解剖剪刀,10厘米,直(WPI,目录号:14393-G)
直镊4.75英寸(Excel,目录号:3-S-SE)
T型针(业务来源,目录号:32351)
秋千式离心机(Thermo Scientific,型号:CL2)
高压灭菌器(Beta Star生命科学,型号:小型消毒器系列26英寸x 26英寸)
II类,A2型生物安全柜(Labconco,目录号:3440009)
恒温水浴(Thermo Fisher Scientific,目录号:TSGP02)
细胞培养箱(Thermo Fisher Scientific,型号:Heracell 240)
14.0 MP数码USB显微镜相机(OMAX显微镜,目录号:A35140U3)
Leica DMI 6000B荧光显微镜(Leica Microsystems,型号:DMI 6000 B)
Lumen 200荧光照明系统(Prior Scientific,型号:Lumen 200)
Coolsnap HQ CCD相机(光度学,型号:Coolsnap HQ)
超级移液器控制器(Corning,目录号:4099)
10μl,200μl,1,000μl移液器(Gilson,型号:Pipetman)
 


软件


 


Photoshop软件(例如,Photoshop CC)
 


程序


 


Prepar 通货膨胀之前隔离
70%乙醇(请参见配方1)。
高压灭菌1.5 ml Eppendorf管。
用70%的乙醇消毒工具。
I型胶原蛋白可覆盖细胞培养板(请参见配方2)。
用基质胶包被细胞培养板(参见配方3)。
板涂
在室温下,在100 mm平板上涂5 ml I型胶原,以完全覆盖平板30分钟。30分钟后,将I型胶原转移到原始试管中,然后风干平板。该板可以存储在A2生物安全柜中,直到可以使用为止。该板将用于预镀。
注意:I型胶原蛋白可以重复使用,以备将来使用。


在室温下,用60 ml的板用3 ml的Matrigel涂膜完全覆盖板20分钟。20分钟后,将Matrigel转移到原始试管中,然后风干板。建议d在Matrigel干燥后立即使用板。该板将用于分化成肌细胞。
注意:Matrigel可以重复使用,以备将来使用。


消化介质(请参见配方4)。
中和介质(请参见配方5)。
生长培养基(见配方6)。
分化培养基(参见配方7)。
0.05%胰蛋白酶(请参见第8条)。
4%多聚甲醛(PFA)固定液(请参见第9条)。
100 mM甘氨酸溶液(请参见配方10)。
阻塞缓冲区(请参见配方11)。
bFGF(请参见第12条)。
PBS-T(请参阅第13条)。
 


肌肉解剖
在实验室中,按照批准的方案牺牲4-6周龄的小鼠。在这种情况下,使用颈脱位。
              注意:无需在A2生物安全柜内进行鼠标解剖。


用70%的乙醇喷洒鼠标,然后将鼠标U钉在解剖板上。
用消毒的剪刀剪掉脚踝处的皮肤。将鼠标置于腹部横向位置时,使用镊子露出股四头肌。用冷PBS收集肌肉。翻转鼠标并将其背面朝上固定。去除下半身的皮肤,解剖两条后肢骨骼肌,然后将它们放入冷的PBS中。
将解剖工具和装有肌肉的平板转移到生物安全柜中。用70%的乙醇擦拭所有解剖工具,以避免来自生物安全柜外部的潜在污染。




D:\重新格式化\ 2020-6-1 \ 1903025--1447 Shihuan Kuang 819878 \视频1.jpg


视频1.小鼠的肌肉解剖。该视频演示了如何隔离后肢肌肉以及如何去除肌肉周围的上皮细胞和脂肪以及肌肉内神经束的详细过程。将肌肉转移到装有PBS的60 mm平板中后,将其在PBS中洗涤数次,用剪刀或剃须刀切碎,并用II型胶原酶消化,以从肌纤维中释放MuSC。


 


肌肉消化
用PBS清洗后肢肌肉,直至无毛。
注意:建议洗几次直到头发完全从肌肉组织上去除。


在薄纸上吸干肌肉。
将肌肉转移到1.5 ml Eppendorf管中。
使用1.5毫升Eppendorf管内的解剖剪刀将肌肉切碎。
注意:细碎的肌肉会产生更多的成肌细胞。


转移剁碎肌成一个15毫升Falcon管。
消化与消化介质肌肉(5毫升,每小鼠)一个37℃的水浴中12分钟,振摇管每2分钟。
用10毫升血清移液器混合消化过的肌肉,直到可以平稳地吸取混合物。
消化在另一个12分钟的37℃水浴中,摇动管每2分钟。
加入5 ml中和培养基停止消化。
在上面放置一个70μm的灭菌的细胞过滤的一个50ml试管中,和预湿用3ml中和介质的过滤器。
注意:预润湿对于细胞顺利通过细胞过滤器很重要。


使用10 ml血清移液器收集含有细胞混合物的培养基,并通过70μm无菌细胞过滤器过滤。
注意:Ç ollect细胞连接,使用1个毫升吸管ltrate下方细胞滤网。


在室温下以2,000 xg 旋转细胞混合物5分钟。
使用真空吸管丢弃上清液。
注意:请勿抽吸细胞沉淀。


用5 ml生长培养基重悬细胞沉淀。
将一只小鼠的细胞播种到未涂覆的100 mm培养板中,并在37°C下孵育4天,并每天在预先存在的培养基上添加5 ml生长培养基,持续3天。
注:细胞混合物和碎纤维的异质群体将在显微镜下被可视化上d 着y 4。


 


原代成肌细胞分离和预铺
上d 着y 4,小纤维片具有圆形主成肌细胞可以看出,和细胞现在准备好进行预镀。
机械分离附着于细胞的通过使用10ml的血清吸液管并转移到移液板的底部一个50ml管中。漂浮细胞也转移到50ml管中。
注意小号:


该方法仅利用机械分离,因此不需要胰蛋白酶。
通过在显微镜下检查,确认大多数细胞是否从板上脱落。
将50 ml试管以2,000 x g 离心5分钟。
注意:在不施加制动的情况下让离心机停止。这通常需要几分钟。


从50毫升试管中吸出培养基。
在仔细并孵育加入3 ml胰蛋白酶到50ml管中重悬细胞一个37℃的水浴中5分钟。
注意:细胞仍然是异质种群。


将50 ml试管以2,000 x g 离心5分钟。
弃去上清液而不干扰沉淀,并用5 ml生长培养基重悬细胞
转移到一个未涂覆的培养板中,孵育37℃的培养箱中45分钟。
45分钟后,将上清液从未包被的培养板转移到胶原包被的板中。
注意:成肌细胞不会在短时间内粘附在培养板上,因此,转移上清液有助于从培养皿底部的其他细胞(如成纤维细胞)中纯化成肌细胞。


可以重复执行步骤D2-D9(预镀)以获得纯度≥95%的成肌细胞。
注意:预镀可以重复两次以增加成肌细胞的纯度。成肌细胞应看起来较小且呈圆形,而成纤维细胞应看起来呈细长形,有时具有双极性过程。


 


培养与差异化
在生长培养基中培养纯化的原代成肌细胞,每2天更换一次培养基。保持细胞密度在80%融合以下,以防止原代成肌细胞融合。
注意:预铺步骤后不需要I型胶原涂层板。


分化之前,在室温下的通风橱中用3 ml Matrigel覆盖60 mm培养板20分钟。
取出Matrigel后,在通风橱中完全蒸发30分钟。
注意:建议不要重复使用Matrigel 5次以上,并且在不使用时应存放在4 °C或冰上。


在室温下用1 ml胰蛋白酶孵育细胞1分钟,以将原代成肌细胞和种子细胞分离到基质胶包被的板上。
注意:将原代成肌细胞在含有4 ml生长培养基的Matrigel包被的板上培养,直到达到所需的细胞密度。


一旦融合达到80-90%,将成肌细胞培养基改为分化培养基。
在接下来的三天内,用分化培养基维持原代成肌细胞。
注意:必须在显微镜下每日监测原代成肌细胞的形态变化。在第3天可以观察到从圆形原代成肌细胞到细长肌管的明显形态学变化。


 


原代成肌细胞的免疫染色
将高压灭菌的盖玻片放在6孔板的孔中。
高压灭菌的盖玻片上的种子成肌细胞
注意小号:


第二天,应在盖玻片上看到附着的成肌细胞。
成肌细胞可以培养数天至所述期望的汇合。
从抽吸生长培养基的培养板中。
通过添加1 ml 4%PFA固定贴壁细胞,并在室温下孵育15分钟。
抽吸4%PFA从所述培养板中。
在室温下用1 ml PBS轻轻清洗3次,每次5分钟。
从培养板上吸出PBS。
加入1 ml 100 mM甘氨酸,在室温下孵育10分钟。
除去100 mM甘氨酸,在室温下用1 ml PBS洗涤5分钟。
重复š 总共3次洗涤TEP 9。
加入1 ml封闭缓冲液,在室温下孵育1-2小时。
加入适量的一抗(Pax7-1:10,MyoD-1:300在封闭缓冲液中)并在4°C下孵育过夜。
在室温下用1ml PBS-T 轻轻清洗3次,每次5分钟。
(山羊抗兔IgG为MF20,DAPI山羊抗小鼠IgG1对的Pax7,MyoG基因,山羊抗小鼠IgG2b为MyoD的添加二级抗体的适当量- :1 1 ,在室温下搅拌1 000在PBS-T) H。
注意:次生胫骨对光敏感,因此以下步骤应在黑暗的房间中执行。


在室温下用1 ml PBS -T 轻轻清洗5次,每次5分钟。
注意:建议多洗手以减少背景污染。


用一滴安装介质安装盖玻片并使其干燥。
在荧光显微镜下评估染色。
 


数据与分析


 


该方案包括在分化前和分化后,用特异性成肌标志物免疫染色后体外观察和评估成肌细胞。通过添加4%PFA固定在生长培养基中培养的原代成肌细胞,并用Pax7和MyoD染色,这是未分化成肌细胞的标志物(图1)。将原代成肌细胞接种到基质胶包被的平板上后,成肌细胞融合度达到80%后,将生长培养基更改为分化培养基。分化3天后,将成肌细胞固定并分别用MyoG和MF20染色,这是成肌分化早期和晚期的标志(图2)。出于研究目的,可用其他抗体对成肌细胞进行染色,但建议与一种成肌标记物共染色以确认成肌细胞的分化状态。


 


D:\重新格式化\ 2020-6-1 \ 1903025--1447 Shihuan Kuang 819878 \ Figs jpg \ 1-word.jpg


图1 。未分化的原代成肌细胞的相衬成像和免疫荧光染色。代表性相衬图像(20x)展示了未分化的原代成肌细胞(A)的形态。细胞培养一天后,将原代成肌细胞固定以进行免疫染色。使用Pax7(红色)和MyoD(绿色)成肌标记物对未分化的原代成肌细胞进行代表性免疫荧光成像。细胞核用DAPI(蓝色)染色(B)。


 


D:\重新格式化\ 2020-6-1 \ 1903025--1447 Shihuan Kuang 819878 \ Figs jpg \ 2-word.jpg


图2 。分化3天后肌管的相衬图像和免疫荧光染色。A. 分化的肌管形态的代表性相衬图像(20x)。B. P rimary成肌细胞上的基质胶包被的平板中培养3天,以形成完全分化的肌管。为了进行免疫染色,将MyoG(红色)和MF20(绿色)用作成肌标记,进行分化以可视化多核肌管。细胞核用DAPI(蓝色)染色。


 


记事簿


 


从幼鼠(3-4周龄)分离的原代成肌细胞的产量高于成年鼠(2个月龄)
当务之急是在解剖和清洗步骤中将头发完全去除,因为多余的头发可能是主要培养物污染的来源。
精细地切成小片小号ö 在切碎步骤f肌肉组织(步骤C 4)是有帮助的成肌细胞增加的产量。
如果成肌细胞的纯度低于95%,则可以进行额外的预镀。
对于染色,可以在室温下将一抗孵育1小时,但强烈建议在4°C下孵育过夜。
 


菜谱


 


70%乙醇
加70毫升100%乙醇和30毫升的DDH 2 ö 。室温保存


I型胶原蛋白涂层
将0.1%的I型胶原蛋白溶于0.1 M乙酸中,并在室温下搅拌混合物1 h,制成10 x 储备液
将10 x I型胶原原液在0.1 M乙酸中稀释至1 x ,以获得0.01%I型胶原
储存在4°C
涂料基质胶
加入1.4体积的DMEM培养基以稀释基质胶


对于2.4毫升溶液,用1.4毫升DMEM培养基稀释1毫升Matrigel,在4°C下储存


消化培养基
通过将分散酶II和II型胶原酶分别溶于浓度为2.4 U / ml 和2.9 U / ml的PBS中制备消化培养基,并补充2.5 mM CaCl 2


对于50 ml的消化介质,d 溶解125 mg分散酶,0.5 g II型胶原酶,然后添加125μl1 M CaCl 2 。储存在-20°C 


中和介质
F-10 Ham培养基中补充了20%FBS和1%青霉素-链霉素。


对于50 ml中和培养基,将在39.5 ml F-10 Ham's培养基中补充10 ml FBS和500μl青霉素-链霉素。储存在4°C


生长培养基
F-10 Ham's培养基补充了20%FBS,4 ng / ml碱性成纤维细胞生长因子和1%青霉素-链霉素


对于50 ml生长培养基,将在F-10 Ham's培养基中补充10 ml FBS,50μlbFGF储备液和500μl青霉素-链霉素。储存在4°C


分化培养基
Dulbecco改良的Eagle培养基(DMEM)补充有2%的马血清和1%的青霉素-链霉素。


对于50 ml分化培养基,DMEM将补充1 ml马血清和500μl青霉素-链霉素。储存在4 °C  


0.05%胰蛋白酶
对于50 ml 0.05%的胰蛋白酶,在40 ml高压灭菌的PBS中加入10 ml的0.25%t胰蛋白酶


储存在4°C


4%多聚甲醛(PFA)
将4%PFA(m / v)溶于PBS溶液,在60℃下搅拌混合物 °C水浴,并通过添加NaOH将pH值提高至7.4,以使PFA粉末更易溶解


100毫升4%PFA,吨AKE 80 毫升PBS溶液,添加4 克对的FA粉末,如上所述溶解PFA,并调整体积至100毫升,PBS溶液中。储存在4 °C


100 mM甘氨酸溶液
将甘氨酸以100 mM 的浓度溶于PBS


对于50 ml的甘氨酸溶液,d 将3.75 g的甘氨酸溶解在50 ml的PBS中。储存在4°C


阻塞缓冲区
PBS补充有5%山羊血清,2%BSA,0.2%Triton X-100、0.1%叠氮化钠


对于100 ml封闭缓冲液,将5 ml山羊血清,2 g BSA,2 ml 10%Triton X-100和0.1 g叠氮化钠添加到PBS中。分装并储存在-20°C


bFGF库存
通过以4:1的浓度在无血清培养基中溶解的bFGF制备的bFGF库存微克/毫升(1 ,000 x)的


对于4μg/ ml bFGF储备液,将4μgbFGF溶于1 ml无血清DMEM中。分装并储存在-20°C


PBS-T
加入PBS中的0.1%Tween-20


对于1升PBS-T溶液1 ml Ť 吐温-20在1升中加入PBS。室温保存


 


致谢


 


该方案改编自我们之前发表在《自然通讯》(Yue 等人,2017)和《生命》(Jia 等人,2019)中的工作。这项工作得到了美国国立卫生研究院(R01AR071649)和美国农业部国家粮食与农业研究所(NC-1184)的资助。


利益争夺


 


作者宣称没有利益冲突。


 


伦理


             


在动物研究中执行的所有程序均由普渡大学的动物护理和使用委员会(PACUC)协议编号1112000440指导。


 


参考资料


 


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贾Z,聂Y,岳F,孔Y,顾丽,加文,TP,刘新,匡光(2019)。小鼠胚胎肌发生和成年肌肉再生中Polo样激酶1的需求。Elife 8:e 47097 。              
Kuang,S.,Kuroda,K.,Le Grand,F.和Rudnicki,MA(2007)。肌肉中卫星干细胞的不对称自我更新和承诺。细胞129(5):999-1010。
Kuang,S.,Gillespie,MA和Rudnicki,MA(2008)。生态位调节肌肉卫星细胞的自我更新和分化。细胞干细胞2(1):22-31。
Liu L.,Cheung,TH,Charville,GW和TA Rando(2015)。通过荧光激活细胞分选法分离骨骼肌干细胞。Nat Protoc 10(10):1612-1624。
N.Motohashi,Y.Asakura和A.Asakura (2014)。肌肉卫星细胞的分离,培养和移植。 Ĵ OVE (86)。DOI:10.3791 / 50846。
马萨诸塞州的鲁德尼基(Rudnicki),F。勒格兰德(Le Grand),I。麦肯内尔(McKinnell)和S. 肌肉干细胞功能的分子调控。冷泉Harb Symp Quant Biol 73:323-331。
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Copyright Kim et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kim, K. H., Qiu, J. and Kuang, S. (2020). Isolation, Culture, and Differentiation of Primary Myoblasts Derived from Muscle Satellite Cells. Bio-protocol 10(14): e3686. DOI: 10.21769/BioProtoc.3686.
  2. Jia, Z., Nie, Y., Yue, F., Kong, Y., Gu, L., Gavin, T. P., Liu, X. and Kuang, S. (2019). A requirement of Polo-like kinase 1 in murine embryonic myogenesis and adult muscle regeneration. Elife 8: e47097.
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