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Isolation, Culture, and Staining of Single Myofibers
单肌纤维的分离、培养和染色   

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Jia Li
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The Journal of Clinical Investigation
Jan 2016

Abstract

Adult skeletal muscle regeneration is orchestrated by a specialized population of adult stem cells called satellite cells, which are localized between the basal lamina and the plasma membrane of myofibers. The process of satellite cell-activation, proliferation, and subsequent differentiation that occurs during muscle regeneration can be recapitulated ex vivo by isolation of single myofibers from skeletal muscles and culturing them under suspension conditions. Here, we describe an improved protocol to evaluate ex vivo satellite cells activation through isolation of single myofibers from extensor digitorum longus (EDL) muscle of mice and culturing and staining of myofiber-associated satellite cells with the markers of self-renewal, proliferation, and differentiation.

Keywords: Single myofiber cultures (单纤维文化), Satellite cells (卫星细胞), Muscle injury (肌肉损伤), Pax7 (Pax7), MyoD (MyoD)

Background

Although skeletal muscle is a fully differentiated post-mitotic tissue, it maintains intrinsic capability to regenerate in response to both genetic and acquired forms of muscle fiber damage (Le Grand and Rudnicki, 2007). Muscle regeneration in adults is mediated by a population of stem cells known as satellite cells which reside between the basal lamina and sarcolemma of myofibers in a mitotically quiescent state (Le Grand and Rudnicki, 2007). In response to muscle trauma, satellite cells become activated and proliferate to produce myoblasts that fuse with pre-existing fibers and with one another to repair or produce new myofibers. A small portion of satellite cells does not differentiate but instead reenters quiescence to maintain the stem cell pool. Satellite cells of all mammalian species express paired-box (Pax) transcription factor Pax7 which is also used as a critical marker to determine the fate of satellite cells in association with other myogenic factors such as MyoD (Le Grand and Rudnicki, 2007; Kuang et al., 2006; Kuang and Rudnicki, 2008).

The in vivo process of muscle regeneration with respect to satellite cell activation, proliferation, and differentiation can be partly recapitulated through suspension culture of myofiber explants (Rosenblatt et al., 1995; Shefer and Yablonka-Reuveni, 2005). The process of myofiber isolation which involves digestion of muscle tissue with matrix-degrading enzymes (e.g., collagenases) and mechanical shearing causes minor trauma which leads to the activation of satellite cells on myofibers. Immediately upon isolation, each myofiber is associated with a fixed number (Pax7+/MyoD-) of satellite cells resting in quiescence. At around 24 h in culture, satellite cells undergo their first round of cell division, through upregulating MyoD (Pax7+/MyoD+) and proliferate to form cell aggregates by 72 h. Satellite cells on cultured myofiber explants can either terminally differentiate (Pax7-/MyoD+) or self-renew (Pax7+/MyoD-) and return to quiescence (Le Grand and Rudnicki, 2007). The myofiber culture system has served as an excellent platform to study the fundamental properties of muscle stem cells and the effects of various genetic manipulations on the basic properties of satellite cells. One of the major advantages of isolated single myofiber cultures is that the physical interaction between the myofiber and satellite cells is preserved in the sense that satellite cells are still maintained beneath the basal lamina (Bischoff, 1986). Furthermore, suspension cultures of myofibers are routinely used to study the effects of various pharmacological compounds and overexpression or knockdown of specific proteins on self-renewal, proliferation, or differentiation of satellite cells (Shefer and Yablonka-Reuveni, 2005; Anderson et al., 2012; Keire et al., 2013). They also provide a useful tool to study the motility of satellite cells on myofibers through live imaging (Siegel et al., 2009). In addition, a few investigators have used isolated single myofibers explants to prepare pure myoblast cultures in which the cells are mostly derived from the myofiber-associated satellite cells.

The isolation of single myofibers from flexor digitorum brevis (FDB) was first described by Bischoff (1986). This protocol was modified later by Rosenblatt et al. to allow handling of single myofibers after collagenase digestion (Rosenblatt et al., 1995). Since then several modifications have been proposed which improved the yield and the handling of the isolated myofibers (Shefer and Yablonka-Reuveni, 2005; Anderson et al., 2012; Verma and Asakura, 2011; Pasut, 2013). However, most of the published protocols still do not produce a sufficient number of myofibers in a single preparation primarily because of under or over digestion with collagenase and the loss of myofibers during picking, sub-culturing, or staining. The amount of time to digest a muscle from rodents may vary with genetic background and healthy versus diseased muscle. In our laboratory, we are now using a six-well tissue culture plate in which the digestion and physical separation of individual myofibers can be visually monitored under a phase contrast microscope. Furthermore, we have developed an improved protocol to stain the myofiber-associated satellite cells for various markers (Hindi et al., 2012; Hindi and Kumar, 2016; Ogura et al., 2013). In this article, we provide the detailed protocol for the isolation, culturing, and staining of myofiber-associated satellite cells for Pax7 and MyoD protein. The same protocol can be adapted for staining of other proteins in satellite cells of myofiber explant.

Materials and Reagents

  1. Disposable borosilicate glass Pasteur pipets (Thermo Fisher Scientific, Fisher Scientific, catalog number: 13-678-20B )
  2. Sterilization pouches (Thermo Fisher Scientific, Fisher Scientific, catalog number: 01-812-51 )
  3. 6-well plates (Corning, Falcon®, catalog number: 353046 )
  4. 0.22 µm filter (EMD Millipore, catalog number: SLGP033RS )
  5. 0.45 µm filter (EMD Millipore, catalog number: SLHV033RS )
  6. 24-well plates (Corning, Falcon®, catalog number: 353047 )
  7. Adult mice (Mus musculus; ≥ 6-week old)
  8. Horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26050-088 )
  9. Dulbecco’s modified Eagle’s medium (DMEM) high glucose, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11995-065 )
  10. N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
  11. Penicillin-streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  12. Collagenase II (Worthington Biochemical Corporation, catalog number: LS004176 )
  13. Ultra-pureTM water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977-015 )
  14. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
  15. 2, 2, 2-tribromoethanol (Avertin) (Sigma-Aldrich, catalog number: T48402 )
  16. 100% ethanol (Thermo Fisher Scientific, Fisher Scientific, catalog number: 2701 )
  17. Tris base (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP152-5 )
  18. Recombinant human fibroblast growth factor-basic (bFGF) (PeproTech, catalog number: 100-18B )
  19. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  20. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437-028 )
  21. Chicken embryo extract (CEE) (Antibody Production Services, catalog number: MD-004E )
  22. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  23. 100% Triton X-100 (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP151-500 )
  24. Glycine (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP381-5 )
  25. 5% (w/v) sodium azide (Thermo Fisher Scientific, Fisher Scientific, catalog number: 71448-16 )
  26. Primary antibody anti-MyoD (rabbit) (Santa Cruz Biotechnology, catalog number: sc-304 )
  27. Primary antibody anti-Pax7 (mouse) (Developmental Studies Hybridoma Bank, catalog number: PAX7 )
  28. Secondary antibody goat anti-rabbit Alexa Fluor® 488 conjugate (Thermo Fisher Scientific, catalog number: A-11034 )
  29. Secondary antibody goat anti-mouse Alexa Fluor® 568 conjugate (Thermo Fisher Scientific, catalog number: A-11004 )
  30. 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, catalog number: D8417 )
  31. Collagenase II (see Recipes)
  32. Digestion medium (see Recipes)
  33. Washing solution (see Recipes)
  34. 70% ethanol (see Recipes)
  35. Basic fibroblast growth factor (bFGF) (see Recipes)
  36. Chicken embryo extract (CEE) (see Recipes)
  37. Myofiber growth medium (MfGM) (see Recipes)
  38. 4% paraformaldehyde (PFA) (see Recipes)
  39. 10% Triton X-100 (see Recipes)
  40. Quenching solution (see Recipes)
  41. Blocking solution (see Recipes)

Equipment

  1. Water bath (Thermo Fisher Scientific, Fisher Scientific, catalog number: 15-462-15Q )
  2. Microscope (Nikon Instruments, model: TE2000 )
  3. CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3578 )

Procedure

  1. Before isolation of myofibers
    1. Borosilicate glass Pasteur pipets ends are too sharp for myofibers. Flame polish them to smooth the end portion and then autoclave (see Note 1).
    2. Autoclave one set of tools containing one small pair of scissors and forceps in a sterilization pouch for extensor digitorum longus (EDL) muscle removal from mouse hind limbs.
    3. Coat 6-well plates with around 1 ml of horse serum (horse serum is used to study satellite cells activation as it keeps myofibers suspended). Let plates sit for 1 min, then remove excess (that you can put back in the original bottle to re-use multiple times, if kept sterile). Avoid bubbles. Leave plates open to dry (drying requires ~20 min). Close plates once they are dried and leave under hood at room temperature till ready to use. These plates will be used for isolation and culturing of myofibers following digestion of EDL muscle with collagenase.
    4. Following the recipe, prepare base of digestion medium (see Note 2).
    5. Following the recipe, prepare collagenase II (see Note 3).
    6. Following the recipe, prepare washing solution. To wash EDL muscle before digestion, prepare a 6-well plate with 3 ml per well of washing solution. Each well is specific for one pair of EDL muscles (right and left). There will be 3 washes per pair of EDL.

  2. Muscle removal and washing
    1. Euthanize mouse by administrating an IP injection of Avertin (2, 2, 2-tribromoethanol) at a dose of 250 mg/kg, followed by cervical dislocation.
    2. Spray 70% ethanol on the whole body and pin the mouse face up.
    3. Shave hair around hind limb area and gently remove the skin using autoclaved scissors and forceps (see Note 4).
    4. Remove carefully fascia surrounding right tibialis anterior (TA) muscle (Video 1).

      Video 1. Step-wise procedure for the isolation of EDL muscle from mouse

    5. Remove carefully TA muscle without damaging underneath EDL muscle (see Note 5). The first muscle removed is TA muscle which can be discarded (Video 1).
    6. Remove carefully EDL muscle (Figure 1). Second muscle removed in Video 1 is EDL muscle. Avoid stretching of muscle during and after isolation (see Notes 5-7).
    7. Put EDL muscle in the well containing washing solution (first wash).
    8. Perform isolation of the other side EDL (try to do this step within next 5 min).
    9. Put EDL muscle in the same well as the other EDL muscle. 


      Figure 1. Isolation of EDL muscle from mice. A. Picture presented here depicts tendon for TA (black arrow) and EDL (green arrow) muscle; B. Mouse hind limb after removal of TA muscle. Arrow points to the tendon of EDL muscle; C. Separating EDL muscle from tendon to tendon; D. Isolation of EDL muscle by cutting distal tendons.

    10. Spray the 6-well plate with 70% ethanol before putting it under the biosafety cabinet.
    11. Perform second and third washes (30 sec each), by transferring EDL muscle to the next well containing washing solution.

  3. Muscle digestion 
    1. Following the recipe, complete the preparation of digestion medium by adding 400 U/ml of collagenase II to the base of digestion medium. Filter the digestion medium with a 0.22 µm filter.
    2. Take a new 6-well plate and put 5 ml of digestion medium per well (for two EDL muscles).
    3. Take autoclaved forceps and remove EDL muscles from the 6-well plate used for washing and put them in the 6-well plate containing digestion medium.
    4. Put the 6-well plate containing EDL muscles for 1.5 h to 2 h in a CO2 incubator set at 37 °C and 5% of CO2. After the first hour of digestion, check the condition of digesting EDL muscles by putting the 6-well plate under an inverted microscope and visualizing if single myofibers are released from the EDL muscle (see Note 8). Using sterile Pasture pipette and digestion medium, gently triturate EDL muscle to accelerate dissociation of single myofibers. Avoid over-digestion in collagenase (Figure 2). Excessive digestion generally results in the isolation of hyper-contracted myofibers (see Note 9).


      Figure 2. Digestion of EDL muscle using collagenase. Representative phase contrast microscopy images presented here show the morphology of good quality EDL muscle at different stages of collagen digestion (top panel). Top-left image shows tightly bundled fibers of EDL muscle prior to digestion. Top-middle image demonstrates partial dissociation of connective tissue at about 45 min of start of collagenase treatment. Top-right image depicts complete digestion and release of myofibers from EDL muscle. Lower panel images demonstrate corresponding phases of muscle digestion in an improperly isolated EDL muscle. Scale bar = 100 µm.

  4. Isolation and culturing of myofibers
    1. Following the recipe, prepare myofiber growth medium (MfGM).
    2. Coat autoclaved Pasteur pipets with horse serum (horse serum prevents myofibers from sticking to glass pipet).
    3. Take a new 6-well plate and put 2 ml of MfGM per well (for neutralization of collagenase activity following EDL muscle digestion).
    4. Remove 6-well plate containing EDL muscle from the incubator. Observe EDL muscle under the microscope to ensure normal structure and shape of single myofibers.
    5. Transfer digested EDL muscles (using autoclaved-flame polished Pasteur pipet) to a new 6-well plate containing 2 ml of MfGM in three wells (see Note 10).
    6. Transfer digested EDL muscles from one well to the next in a serial manner with autoclaved Pasteur pipet. Allow digested EDL’s to set in each of the 3 wells for 30 sec. This will neutralize the collagenase enzymatic activity and stop the digestion process.
    7. Take 6-well plates coated previously with horse serum and put 3 ml of MfGM per well (for culturing of myofibers).
    8. Transfer neutralized EDL muscles in the coated 6-well plate containing MfGM (i.e., well #1). Using autoclaved coated Pasture pipet, gently flush EDL muscle and myofiber bundles with MfGM. Do not vigorously triturate the muscle as it can cause damage to myofibers (see Note 11).
    9. Transfer the EDL muscles from well #1 to well #2. Flush gently EDL with autoclaved coated Pasteur pipet.
    10. Move the EDL muscles from well #2 to well #3 and repeat the same process until well #6. The goal is to have myofibers from EDL muscles in each well. Representative images of isolated single myofibers are presented in Figure 3.


      Figure 3. Images of normal and hypercontracted isolated single myofiber cultures. Left image represents isolated single myofibers following a successful isolation. Fibers are mostly straight and translucent and their surface is clear of any shears or tears. Right image shows fiber morphology following an inadequate preparation. Red arrows point to hypercontracted fibers while blue arrows point to fragmented fibers. Scale bar = 100 µm.

    11. Observe EDL myofibers under the microscope for their shape and structure (see Note 12).
    12. Put the 6-well plate containing EDL myofibers in a CO2 incubator set at 37 °C and 5% of CO2. If analysis of satellite cells is desired in the quiescent stage, fix myofibers immediately and proceed to staining. For studying activation of satellite cells, culture myofibers for desired time points. Normally, large satellite cell clusters are observed at 72 h post isolation.

  5. Immunostaining of satellite cells for Pax7 and MyoD protein
    1. With a pipette, carefully remove 2 ml of MfGM, leaving remaining 1 ml with myofibers (see Note 13).
    2. Fix myofibers by adding 1 ml of 4% PFA to each well containing myofibers and incubate for 5 min at room temperature (RT).
    3. With a pipette, carefully remove MfGM and 4% PFA, leaving myofibers inside the well.
    4. Add 1 ml of 4% PFA to each well. Incubate the plates for 10 min at RT.
    5. Carefully remove 4% PFA solution while leaving myofibers inside the well.
    6. Wash each well containing myofibers with 1 ml of PBS. Incubate for 5 min at RT. Carefully remove PBS leaving myofibers inside the well. Repeat this step 2 times (2 x 5 min).
    7. Prepare quenching solution.
    8. Add 2 ml of quenching solution to each well. Incubate for 7 to 10 min at RT.
    9. Carefully remove quenching solution, leaving myofibers inside the well.
    10. Wash wells with 1 ml of PBS. Incubate for 5 min at RT. Remove PBS and leave myofibers inside the well. Repeat this step 3 times (3 x 5 min).
    11. Prepare blocking solution.
    12. Add 1 ml of blocking solution to each well. Incubate for 60 min at RT.
    13. Using Pasture pipet, transfer myofibers from the 6-well plate to a 24-well plate (to minimize the quantity of reagents used).
    14. Make 1:100 dilution of MyoD antibody in blocking solution. Similarly, make 1:5 dilution of Pax7 antibody in blocking solution.
    15. Carefully remove blocking solution while leaving myofibers inside the well.
    16. Add 100 µl of each diluted antibody solution per well of the 24-well plate containing myofibers. After this, the final dilution of anti-MyoD will be 1:200 and anti-Pax7 will be 1:10. Incubate the plate overnight at 4 °C.
    17. Next day, remove primary antibody solution from each well while leaving myofibers inside the well.
    18. Add 200 μl of PBS to each well of the 24-well plate for washing myofibers. Incubate for 5 min at RT. Remove PBS from the wells and add fresh PBS. Repeat this step 3 times (3 x 5 min).
    19. Make 1:1,500 dilution of two secondary antibody (i.e., goat anti-rabbit Alexa Fluor 488 conjugate and goat anti-mouse Alexa Fluor 568 conjugate) in blocking solution.
    20. Add 200 µl of secondary antibodies solution to the well. Incubate for 60 min at RT in the dark.
    21. Remove secondary antibodies from each well while leaving myofibers inside.
    22. Add 200 μl of PBS to each well and incubate for 5 min at RT. Carefully remove PBS and leave the myofibers inside the well. Repeat this step 3 times (3 x 5 min).
    23. Make 1:5,000 dilution of DAPI in PBS.
    24. Add 200 µl of diluted DAPI solution to each well. Incubate for 3 min at RT in the dark.
    25. Remove DAPI from each well, leaving myofibers inside.
    26. Add 200 μl of PBS to each well. Incubate for 5 min at RT. Remove PBS while leaving myofibers inside the well. Repeat this step 2 times (2 x 5 min).
    27. Finally, add 200 μl of PBS to each well.
    28. Analyze the myofiber-associated satellite cells for the expression of MyoD (green) and Pax7 (red) under a fluorescent microscope. Representative individual anti-MyoD, anti-Pax7, DAPI stained and merged images generated using this protocol have been published in our recent publications (Hindi and Kumar, 2016; Ogura et al., 2015). A representative image of staining of satellite cell cluster on myofibers after 72 h of culturing is presented in Figure 4.


      Figure 4. Staining of myofiber-associated satellite cells for Pax7 and MyoD proteins. Representative individual and merged images of myofiber-associated satellite cell cluster stained with anti-Pax7, anti-MyoD, and DAPI following 72 h of culturing. Scale bar = 100 µm.

Data analysis

For individual experiments, the sample size should be determined by power analysis. For most of our studies, we have used from 3-5 mice in each group. For analysis of Pax7+ or MyoD+ cells, it is highly recommended that 18-22 myofibers are analyzed from each EDL muscle of each mouse. Data are collected as average number of Pax7+/MyoD-, Pax7+/MyoD+, and Pax7-/MyoD+ cells on each myofibers. These numbers are used to calculate the average from different mice in each group. We present the data as mean ± standard deviation (SD). We used paired or unpaired Student’s t-test to determine statistical differences among different groups similar to as described (Hindi and Kumar, 2016; Ogura et al., 2015; Ogura et al., 2013). A P < 0.05 is considered as statistically significant.

Notes

  1. It is important to use glass Pasteur pipets in which their ends are smoothened by flame. Sharp end Pasteur pipets can damage the myofibers during picking and handling in subsequent steps.
  2. Do not add collagenase at this point to preserve enzymatic activity.
  3. Collagenase aliquots can be prepared ahead of time and stored at -20 °C.
  4. It is critical to use sterile surgical tools to avoid contamination of cultures especially for long-term analysis.
  5. Make sure that sharp side of scissors is not facing muscle surface as this could shear and damage the muscle.
  6. In order to obtain undamaged myofibers, it is necessary to isolate EDL muscle from tendon to tendon during muscle removal process.
  7. When handling EDL muscle at any step, make sure to hold it by its distal tendons and avoid pinching or grabbing it from the mid-belly.
  8. Attempting to triturate or release myofibers from EDL before muscle is adequately digested will subject myofibers to mechanical damage because greater trituration force will be required which will reduce the yield of healthy myofibers.
  9. EDL muscle from wild-type C57BL6 mice get digested within 2 h. However, some other strains may take longer or shorter time for sufficient digestion. It is highly recommended to monitor the digestion of muscle under microscope. Under- or over-digestion of muscle will lead to death and poor yield of myofibers during subsequent steps.
  10. At this step, do not allow the digested muscle to be aspirated into the Pasture pipet as this could damage the myofibers.
  11. Once some myofibers have been released into the medium in one well, do not continue the trituration process in the same well to release more myofibers, as previously released myofibers may be damaged during the attempt to dissociate more myofibers from the muscle. For that reason, immediately transfer the muscle to the next well of the tissue culture plate and continue the trituration process to release more myofibers. Continue this process till maximum number of myofibers can be released.
  12. Healthy myofibers should look translucent with no signs of damage or shearing. Regular striations can be observed on the surface of myofibers under high magnification.
  13. Removing the entire growth medium before fixing the myofibers may result in bending or shrinking of myofibers, therefore it is advisable to add 4% PFA to myofibers while still in residual amount of growth medium to maintain morphological and structural integrity.

Recipes

  1. Collagenase II
    Prepare 4,000 U/ml stock in ultra-pure water.
  2. Digestion medium
    1. Prepare base of digestion medium, by supplementing DMEM with 2.5% HEPES and 1% Penicillin (Pen)/streptomycin (Strep) solution. Keep at 37 °C in a water bath till ready for use.
    2. Once muscle has been isolated and ready for digestion, add collagenase II to make a final concentration of 400 U/ml. Shake briefly by hand.
    3. Before use, filter digestion medium using a 0.22 µm filter.
      Note: For two EDL muscles prepare 5 ml of digestion medium.
  3. Washing solution
    PBS with 1% Pen/Strep
  4. 70% ethanol
    Combine 700 ml of 100% ethanol with 300 ml of deionized water.
  5. Basic fibroblast growth factor (bFGF)
    Dilute 50 µg of bFGF (50 µg/ml) in 1 ml of 5 mM Tris (pH 7.6) with 0.1% BSA.
  6. Chicken embryo extract (CEE)
    Add 10 ml of PBS in a bottle containing lyophilized CEE (50 µg/ml).
  7. Myofiber growth medium (MfGM)
    Supplement DMEM with 10% FBS, 1% Pen/Strep, 2% CEE, and 10 ng/ml bFGF.
    Warm MfGM at 37 °C in a water bath.
    Before use, filter MfGM with a 0.22 µm filter.
    Note: For two EDL muscles prepare 36 ml of MfGM.
  8. 4% paraformaldehyde (PFA)
    Dilute 0.4 g of PFA in 10 ml of PBS.
    Shake vigorously at 60 °C and 250 rpm for 1-2 h until completely dissolved.
    Before use, filter 4% PFA with a 0.45 µm filter.
  9. 10% Triton X-100
    Combine 100 µl of 100% Triton X-100 with 900 µl of PBS.
  10. Quenching solution
    For 10 ml solution, combine 75.07 mg of glycine with 200 µl of 10% Triton X-100 and 50 µl of 5% sodium azide.
    Complete to 10 ml with PBS.
  11. Blocking solution
    For 10 ml solution, combine 200 mg of BSA with 200 µl of 10% Triton X-100 and 500 µl of horse serum.
    Complete to 10 ml with PBS.
    Before use, filter blocking solution with a 0.45 µm filter.

Acknowledgments

This work was supported by funding from NIH grants AR059810, AR068313, and AG029623 (to A. Kumar) and AR069985 (to S. M. Hindi).

References

  1. Anderson, J. E., Wozniak, A. C. and Mizunoya, W. (2012). Single muscle-fiber isolation and culture for cellular, molecular, pharmacological, and evolutionary studies. Methods Mol Biol 798: 85-102.
  2. Bischoff, R. (1986). Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115(1): 129-139.
  3. Hindi, S. M., Paul, P. K., Dahiya, S., Mishra, V., Bhatnagar, S., Kuang, S., Choi, Y. and Kumar, A. (2012). Reciprocal interaction between TRAF6 and notch signaling regulates adult myofiber regeneration upon injury. Mol Cell Biol 32(23): 4833-4845.
  4. Hindi, S. M. and Kumar, A. (2016). TRAF6 regulates satellite stem cell self-renewal and function during regenerative myogenesis. J Clin Invest 126(1): 151-168.
  5. Kuang, S., Charge, S. B., Seale, P., Huh, M. and Rudnicki, M. A. (2006). Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis. J Cell Biol 172(1): 103-113.
  6. Kuang, S. and Rudnicki, M. A. (2008). The emerging biology of satellite cells and their therapeutic potential. Trends Mol Med 14(2): 82-91.
  7. Keire, P., Shearer, A., Shefer, G. and Yablonka-Reuveni, Z. (2013). Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol 946: 431-468.
  8. Le Grand, F. and Rudnicki, M. A. (2007). Skeletal muscle satellite cells and adult myogenesis. Curr Opin Cell Biol 19(6): 628-633.
  9. Ogura, Y., Hindi, S. M., Sato, S., Xiong, G., Akira, S. and Kumar, A. (2015). TAK1 modulates satellite stem cell homeostasis and skeletal muscle repair. Nat Commun 6: 10123.
  10. Ogura, Y., Mishra, V., Hindi, S. M., Kuang, S. and Kumar, A. (2013). Proinflammatory cytokine tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) suppresses satellite cell self-renewal through inversely modulating Notch and NF-κB signaling pathways. J Biol Chem 288(49): 35159-35169.
  11. Pasut, A., Jones, A. E. and Rudnicki, M. A. (2013). Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle. J Vis Exp(73): e50074.
  12. Rosenblatt, J. D., Lunt, A. I., Parry, D. J. and Partridge, T. A. (1995). Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 31(10): 773-779.
  13. 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.
  14. Siegel, A. L., Atchison, K., Fisher, K. E., Davis, G. E. and Cornelison, D. D. (2009). 3D timelapse analysis of muscle satellite cell motility. Stem Cells 27(10): 2527-2538.
  15. Verma, M. and Asakura, A. (2011). Efficient single muscle fiber isolation from alcohol-fixed adult muscle following beta-galactosidase staining for satellite cell detection. J Histochem Cytochem 59(1): 60-67.

简介

成年骨骼肌再生由被称为卫星细胞的成人干细胞的专门群体协调,其被定位在基底层和肌纤维的质膜之间。在肌肉再生期间发生的卫星细胞激活,增殖和随后分化的过程可以通过从骨骼肌中分离单个肌纤维并在悬浮条件下培养它们而在体外重现。在这里,我们描述了一种改进的协议,通过从小鼠的伸肌腱长肌(EDL)肌肉中分离单个肌纤维来评价离体细胞的活化,并培养和染色肌纤维相关的卫星细胞的标记物自我更新,增殖和分化。

[背景] 虽然骨骼肌是一种完全分化的有丝分裂后组织,但是它保持了内在的能力,以对遗传和获得性肌肉纤维损伤的形式Grand和Rudnicki,2007)。成年人的肌肉再生由称为卫星细胞的干细胞群体介导,所述细胞位于有丝分裂静止状态的肌纤维的基底层和肌纤维膜之间(Le Grand和Rudnicki,2007)。响应于肌肉创伤,卫星细胞变得活化和增殖以产生与预先存在的纤维融合的成肌细胞,并彼此相互修复或产生新的肌纤维。一小部分卫星细胞不分化,而是重新进入静止以维持干细胞库。所有哺乳动物物种的卫星细胞表达配对盒(Pax)转录因子Pax7,其也用作确定与其他生肌因子如MyoD相关的卫星细胞命运的关键标记(Le Grand和Rudnicki,2007; et al。,2006; Kuang and Rudnicki,2008)。
  可以通过肌纤维外植体的悬浮培养部分地重现关于卫星细胞活化,增殖和分化的肌肉再生的体内过程(Rosenblatt等人,1995; Shefer和Yablonka-Reuveni,2005)。肌纤维分离的过程涉及用基质降解酶(例如胶原酶)和机械剪切消化肌肉组织,导致轻微的创伤,导致卫星细胞在肌纤维上的活化。在分离后,每个肌纤维立即与静止的静止的卫星细胞的固定数目(Pax7 + /MyoD - )相关。在培养物中约24小时时,卫星细胞通过上调MyoD(Pax7 + sup/+/MyoD + )进行其第一轮细胞分裂,并通过72小时增殖形成细胞聚集。培养的肌纤维外植体上的卫星细胞可以终末分化(Pax7 /MyoD + )或自我更新(Pax7 + /MyoD - ),并恢复静止(Le Grand和Rudnicki,2007)。肌纤维培养系统作为一个极好的平台,研究肌肉干细胞的基本性质和各种遗传操作对卫星细胞的基本性质的影响。分离的单个肌纤维培养物的主要优点之一是肌纤维和卫星细胞之间的物理相互作用被保持在卫星细胞仍然保持在基底层下方的意义上(Bischoff,1986)。此外,肌纤维的悬浮培养物常规用于研究各种药理化合物的作用和特异性蛋白质对卫星细胞的自我更新,增殖或分化的过表达或敲低(Shefer和Yablonka-Reuveni,2005; Anderson等al ,2012; Keire ,。,2013)。它们还提供了用于通过实时成像研究卫星细胞对肌纤维的运动性的有用工具(Siegel等人,2009)。此外,一些研究人员使用单独的单个肌纤维外植体来制备纯成肌细胞培养物,其中细胞主要来源于与肌纤维相关的卫星细胞。
  Bischoff(1986)首次描述了从屈肌小疱(FDB)分离单肌纤维。该协议稍后由Rosenblatt等人修改。以允许在胶原酶消化后处理单个肌纤维(Rosenblatt等人,1995)。自从那时以来,已经提出了改进分离的肌纤维的产量和处理的几种修饰(Shefer和Yablonka-Reuveni,2005; Anderson等人,2012; Verma和Asakura, )。然而,大多数公开的方案仍然不能在单一制剂中产生足够数量的肌纤维,主要是因为胶原酶的消化或过度消化以及在挑选,亚培养或染色期间肌纤维的损失。从啮齿动物消化肌肉的时间量可以变化w

关键字:单纤维文化, 卫星细胞, 肌肉损伤, Pax7, MyoD

材料和试剂

  1. 一次性硼硅酸盐玻璃巴斯德吸管(Thermo Fisher Scientific,Fisher Scientific,目录号:13-678-20B)
  2. 灭菌袋(Thermo Fisher Scientific,Fisher Scientific,目录号:01-812-51)
  3. 6孔板(Corning,Falcon ,目录号:353046)
  4. 0.22μm过滤器(EMD Millipore,目录号:SLGP033RS)
  5. 0.45μm过滤器(EMD Millipore,目录号:SLHV033RS)
  6. 24孔板(Corning,Falcon ,目录号:353047)
  7. 成年小鼠(Mus musculus;≥6周龄)
  8. 马血清(Thermo Fisher Scientific,Gibco TM ,目录号:26050-088)
  9. Dulbecco改良的Eagle培养基(DMEM)高葡萄糖,丙酮酸盐(Thermo Fisher Scientific,Gibco TM,目录号:11995-065)
  10. N-2-羟乙基哌嗪-N-2-乙烷磺酸(HEPES)(1μM)(Thermo Fisher Scientific,Gibco TM,目录号:15630-080)
  11. 青霉素 - 链霉素(Pen/Strep)(Thermo Fisher Scientific,Gibco TM ,目录号:15140-122)
  12. 胶原酶II(Worthington Biochemical Corporation,目录号:LS004176)
  13. 超纯水(Thermo Fisher Scientific,Invitrogen TM ,目录号:10977-015)
  14. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10010-023)
  15. 2,2,2-三溴乙醇(Avertin)(Sigma-Aldrich,目录号:T48402)。
  16. 100%乙醇(Thermo Fisher Scientific,Fisher Scientific,目录号:2701)
  17. Tris碱(Thermo Fisher Scientific,Fisher Scientific,目录号:BP152-5)
  18. 重组人成纤维细胞生长因子 - 碱性(bFGF)(PeproTech,目录号:100-18B)
  19. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  20. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10437-028)
  21. 鸡胚提取物(CEE)(抗体生产服务,目录号:MD-004E)
  22. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  23. 100%Triton X-100(Thermo Fisher Scientific,Fisher Scientific,目录号:BP151-500)
  24. 甘氨酸(Thermo Fisher Scientific,Fisher Scientific,目录号:BP381-5)
  25. 5%(w/v)叠氮化钠(Thermo Fisher Scientific,Fisher Scientific,目录号:71448-16)
  26. 第一抗体抗MyoD(兔)(Santa Cruz Biotechnology,目录号:sc-304)
  27. 初级抗体抗Pax7(小鼠)(Developmental Studies Hybridoma Bank,目录号:PAX7)
  28. 第二抗体山羊抗兔Alexa Fluor 488缀合物(Thermo Fisher Scientific,目录号:A-11034)
  29. 第二抗体山羊抗小鼠Alexa Fluor 568缀合物(Thermo Fisher Scientific,目录号:A-11004)
  30. 4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)(Sigma-Aldrich,目录号:D8417)
  31. 胶原酶II(参见配方)
  32. 消化介质(参见配方)
  33. 洗涤液(见配方)
  34. 70%乙醇(见配方)
  35. 碱性成纤维细胞生长因子(bFGF)(参见配方)
  36. 鸡胚提取物(CEE)(参见食谱)
  37. 肌纤维生长培养基(MfGM)(参见配方)
  38. 4%多聚甲醛(PFA)(参见配方)
  39. 10%Triton X-100(参见配方)
  40. 淬火溶液(参见配方)
  41. 阻止解决方案(参见配方)

设备

  1. 水浴(Thermo Fisher Scientific,Fisher Scientific,目录号:15-462-15Q)
  2. 显微镜(Nikon Instruments,型号:TE2000)
  3. CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3578)。

程序

  1. 在隔离肌纤维之前
    1. 硼硅酸盐玻璃巴斯德移液管末端对于肌纤维来说太锋利。 火焰抛光使端部平滑,然后高压灭菌(见注1)
    2. 在灭菌袋中高压灭菌一套包含一把小剪刀和镊子的工具,用于从小鼠后肢取出长伸肌(EDL)肌肉。
    3. 用约1ml马血清(马血清用于研究卫星细胞活化,因为它保持肌纤维悬浮)涂覆6孔板。让板放置1分钟,然后除去多余的(你可以放回原来的瓶子重复使用多次,如果保持无菌)。避免气泡。将板开放干燥(干燥需要〜20分钟)。关闭板一旦它们被干燥并且在室温下在通风橱下留下,直到准备使用。这些板将用于在用胶原酶消化EDL肌肉后分离和培养肌纤维。
    4. 按照配方,准备消解培养基(见注2)
    5. 按照食谱,准备胶原酶II(见注3)
    6. 按照配方,准备洗涤溶液。为了在消化前洗涤EDL肌肉,制备6孔板,每孔3ml的洗涤溶液。每个孔对于一对EDL肌肉(右和左)是特异性的。每对EDL将有3次洗涤
  2. 肌肉去除和洗涤
    1. 通过以250mg/kg的剂量IP注射Avertin(2,2,2-三溴乙醇),然后颈脱位来安乐死小鼠。
    2. 在整个身体上喷洒70%乙醇,并将小鼠面朝上。
    3. 在后肢区域剃刮头发,并使用高压灭菌剪刀和镊子轻轻取出皮肤(见注4)。
    4. 小心移除围绕右胫前肌(TA)肌肉的筋膜(视频1)
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    5. 小心去除TA肌肉,而不损伤EDL肌肉下面(见注5)。 第一个去除的肌肉是可以丢弃的TA肌肉(视频1)
    6. 仔细去除EDL肌肉(图1)。 视频1中去除的第二肌肉是EDL肌肉。 避免在隔离期间和隔离后拉伸肌肉(见注释5-7)。
    7. 将EDL肌肉放入含有洗涤溶液的孔中(第一次洗涤)
    8. 执行另一侧EDL的隔离(尝试在接下来的5分钟内执行此步骤)。
    9. 将EDL肌肉放在与其他EDL肌肉相同的井中。


      图1.来自小鼠的EDL肌肉的分离 A.这里呈现的图片描绘了TA(黑色箭头)和EDL(绿色箭头)肌肉的腱; B.移除TA肌肉后的小鼠后肢。 箭头指向EDL肌肉的腱; 将EDL肌肉从腱分离为腱; D.通过切割远端肌腱来分离EDL肌肉。

    10. 将6孔板用70%乙醇喷洒在生物安全柜下。
    11. 进行第二次和第三次洗涤(每次30秒),通过将EDL肌肉转移到包含洗涤溶液的下一个孔
  3. 肌肉消化
    1. 根据配方,通过添加400 U/ml胶原酶II到消化培养基的基础完成消化培养基的制备。用0.22μm过滤器过滤消解介质。
    2. 取一个新的6孔板,每孔加入5毫升消化培养基(两个EDL肌肉)
    3. 取高压灭菌镊子,从用于洗涤的6孔板中清除EDL肌肉,并将其放入含有消化培养基的6孔板中。
    4. 将含有EDL肌肉的6孔板在设定为37℃和5%CO 2的CO 2培养箱中放置1.5小时至2小时。在消化第一小时后,通过将6孔板置于倒置显微镜下并观察是否单个肌纤维从EDL肌肉释放,检查消化EDL肌肉的状况(参见注释8)。使用无菌牧场移液管和消化介质,轻轻研磨EDL肌肉加速单肌纤维的解离。避免过度消化胶原酶(图2)。过度消化通常导致高收缩肌纤维的分离(见注9)。


      图2.使用胶原酶消化EDL肌肉。本文提供的代表性相差显微镜图像显示了在不同胶原消化阶段(上图)的良好质量EDL肌肉的形态。左上图显示了EDL肌肉在消化前的紧密捆束纤维。上中图像显示在胶原酶处理开始约45分钟时结缔组织的部分解离。右上图描述了从EDL肌肉完全消化和释放肌纤维。下图显示了在不适当分离的EDL肌肉中肌肉消化的相应阶段。比例尺=100μm。

  4. 肌纤维的分离和培养
    1. 按照食谱,准备肌纤维生长培养基(MfGM)
    2. 用马血清(马血清防止肌纤维粘在玻璃吸管)涂覆高压灭菌的巴斯德吸管。
    3. 取一个新的6孔板,每孔放2毫升MfGM(EDL肌肉消化后中和胶原酶活性)。
    4. 从培养箱中取出含有EDL肌肉的6孔板。在显微镜下观察EDL肌肉,以确保单肌纤维的正常结构和形状
    5. 转移消化的EDL肌肉(使用高压灭菌火焰抛光的巴斯德吸管)到一个新的6孔板,包含2毫升MfGM在三个井(见注10)。
    6. 转移消化的EDL肌肉从一个井到下一个连续的方式用高压灭菌的巴斯德吸管。允许消化的EDL's设置在每个3个孔中30秒。这将中和胶原酶酶活性并停止消化过程。
    7. 取先前用马血清包被的6孔板,并每孔加入3ml MfGM(用于培养肌纤维)。
    8. 在包含MfGM(即,孔#1)的包被的6孔板中转移中和的EDL肌肉。使用高压灭菌包衣牧场吸管,轻轻冲洗EDL肌肉和肌纤维束与MfGM。不要剧烈磨损肌肉,因为它可能导致肌纤维损伤(见注11)。
    9. 将EDL肌肉从#1井转移到#2井。用高压灭菌的涂覆的巴斯德吸液管轻轻地冲洗EDL
    10. 将EDL肌肉从井#2移动到井#3,并重复相同的过程,直到井#6。目标是在每个井中使用来自EDL肌肉的肌纤维。孤立单肌纤维的代表性图像如图3所示。


      图3.正常和超收缩的单独单肌纤维培养物的图像。左图表示成功分离后的孤立单肌纤维。纤维大多是直的和半透明的,它们的表面没有任何剪切或撕裂。右图显示了不充分制备后的纤维形态。红色箭头指向超收缩纤维,而蓝色箭头指向断裂纤维。比例尺=100μm。

    11. 在显微镜下观察EDL肌纤维的形状和结构(见注12)
    12. 将含有EDL肌纤维的6孔板置于设定在37℃和5%CO 2的CO 2培养箱中。如果需要在静止期分析卫星细胞,立即固定肌纤维并进行染色。为了研究卫星细胞的激活,培养肌纤维达到所需的时间点。通常,在分离后72小时观察到大的卫星细胞簇
  5. 卫星细胞对Pax7和MyoD蛋白的免疫染色
    1. 用移液管,小心地删除2毫升MfGM,留下1毫升与肌纤维(见注13)。
    2. 通过加入1毫升4%PFA到每个含有肌纤维的孔中并在室温(RT)下孵育5分钟来固定肌纤维。
    3. 用移液管,小心地去除MfGM和4%PFA,将肌纤维留在孔中。
    4. 向每个孔中加入1ml的4%PFA。 在RT孵育平板10分钟。
    5. 小心地清除4%PFA溶液,同时将光纤放在孔内。
    6. 用1ml PBS洗涤含有肌纤维的每个孔。 在室温下孵育5分钟。 小心取出PBS离开孔内的肌纤维。 重复此步骤2次(2 x 5分钟)。
    7. 准备淬火溶液。
    8. 向每个孔中加入2ml淬灭溶液。 在室温下孵育7至10分钟。
    9. 小心地清除淬火溶液,将肌纤维留在井内
    10. 用1ml PBS洗孔。 在室温下孵育5分钟。 取出PBS,离开肌纤维在井内。 重复此步骤3次(3 x 5分钟)。
    11. 准备封闭溶液。
    12. 向每个孔中加入1ml封闭溶液。 在室温下孵育60分钟。
    13. 使用牧场移液管,将肌纤维从6孔板转移到24孔板(以使所用试剂的量最小化)。
    14. 在封闭液中制成1:100稀释的MyoD抗体。类似地,在封闭溶液中制备Pax7抗体的1:5稀释液。
    15. 小心地除去阻塞溶液,同时将光纤放在孔内。
    16. 加入100微升每个稀释的抗体溶液每孔的24孔板含有肌纤维。之后,抗MyoD的最终稀释度为1:200,抗Pax7将为1:10。将板在4℃下孵育过夜。
    17. 第二天,从每个孔中移除初级抗体溶液,同时将肌纤维留在孔内
    18. 在24孔板的每个孔中加入200μlPBS以洗涤肌纤维。在室温下孵育5分钟。从孔中取出PBS,加入新鲜的PBS。重复此步骤3次(3 x 5分钟)。
    19. 在封闭溶液中制备1:1,500稀释的两种第二抗体(即山羊抗兔Alexa Fluor 488缀合物和山羊抗小鼠Alexa Fluor 568缀合物)。
    20. 在孔中加入200μl次级抗体溶液。 在室温下避光孵育60分钟。
    21. 从每个孔中移除二抗,同时留下肌纤维
    22. 在每个孔中加入200μlPBS,并在室温下孵育5分钟。 小心取出PBS,并留在井内的肌纤维。 重复此步骤3次(3 x 5分钟)。
    23. 制备1:5,000稀释的DAPI在PBS中
    24. 在每个孔中加入200μl稀释的DAPI溶液。 在室温下避光孵育3分钟。
    25. 从每个井中取出DAPI,留下肌纤维内。
    26. 在每个孔中加入200μlPBS。在室温下孵育5分钟。取出PBS,同时将肌纤维留在孔内。重复此步骤2次(2 x 5分钟)。
    27. 最后,在每个孔中加入200μlPBS
    28. 分析肌纤维相关卫星细胞表达的MyoD(绿色)和Pax7(红色)在荧光显微镜下。使用该方案产生的代表性个体抗MyoD,抗Pax7,DAPI染色和合并图像已经在我们最近的出版物中发表(Hindi和Kumar,2016; Ogura等人,2015)。培养72小时后卫星细胞簇在肌纤维上染色的代表性图像示于图4中

      图4.对于Pax7和MyoD蛋白质的肌纤维相关卫星细胞的染色。在72小时后,用抗Pax7,抗MyoD和DAPI染色的肌纤维相关卫星细胞簇的代表性个体和合并图像的培养。比例尺=100μm。

数据分析

对于单个实验,样品大小应通过功率分析确定。对于我们的大多数研究,我们使用每组3-5只小鼠。对于Pax7 + 或MyoD + 细胞的分析,强烈建议从每只小鼠的每个EDL肌肉分析18-22个肌纤维。数据作为Pax7 /MyoD - ,Pax7 + /MyoD + 的平均数收集,Pax7 < sup> - /MyoD + 细胞。这些数字用于计算每组中不同小鼠的平均值。我们将数据表示为平均值±标准差(SD)。我们使用配对或不配对的学生检验来确定不同组之间的统计差异,类似于所述(Hindi和Kumar,2016; Ogura等人,2015; Ogura等人, em>等人,2013)。 A 0.05被认为具有统计学意义。

笔记

  1. 重要的是使用玻璃巴斯德移液管,其中它们的末端被火焰平滑。尖端巴斯德吸管在后续步骤中的拾取和处理过程中可能损坏肌纤维。
  2. 此时不要添加胶原酶以保持酶活性。
  3. 胶原酶等分试样可以提前制备并储存在-20℃
  4. 使用无菌手术工具以避免培养物的污染,特别是长期分析是至关重要的
  5. 确保剪刀的锋利边不面对肌肉表面,因为这可能剪切和损坏肌肉
  6. 为了获得未受损的肌纤维,有必要在肌肉去除过程中将EDL肌肉从腱分离为腱。
  7. 在任何步骤处理EDL肌肉时,请确保通过其远端肌腱保持它,避免挤压或从中腹部抓住它。
  8. 试图在肌肉充分消化之前从EDL中研磨或释放肌纤维将使肌纤维受到机械损伤,因为需要更大的研磨力,这将降低健康肌纤维的产量。
  9. 来自野生型C57BL6小鼠的EDL肌肉在2小时内消化。然而,一些其他菌株可能需要更长或更短的时间来进行充分消化。强烈建议在显微镜下监测肌肉的消化。肌肉的过度或过度消化将导致在随后的步骤期间肌细胞的死亡和较差的产量。
  10. 在这一步,不要让消化的肌肉吸入牧场移液管,因为这可能会损坏肌纤维。
  11. 一旦一些肌纤维已经释放到一个孔中的培养基中,不要在同一个孔中继续研磨过程以释放更多的肌纤维,因为先前释放的肌纤维可能在试图从肌肉解离更多肌纤维的过程中被损坏。为此,立即将肌肉转移到组织培养板的下一个孔,并继续研磨过程以释放更多的肌纤维。继续此过程,直到可以释放最大数量的肌纤维
  12. 健康肌纤维应该看起来半透明,没有损伤或剪切的迹象。在高放大倍率下可以在肌纤维的表面上观察到规则的条纹
  13. 在固定肌纤维之前除去整个生长培养基可能导致肌纤维的弯曲或收缩,因此建议向肌纤维中加入4%PFA,同时仍然在剩余量的生长培养基中以保持形态和结构完整性。

食谱

  1. 胶原酶II 在超纯水中制备4,000 U/ml原液
  2. 消化培养基
    1. 通过用2.5%HEPES和1%青霉素(Pen)/链霉素(Strep)溶液补充DMEM制备消化培养基的底物。 保持在37°C在水浴中,直到准备好 使用。
    2. 一旦肌肉已被分离并准备消化,添加胶原酶II,使最终浓度为400 U/ml。 用手轻轻摇动。
    3. 使用前,使用0.22μm过滤器过滤消解介质 注意:对于两个EDL肌肉,准备5ml消化介质。
  3. 洗涤溶液
    PBS,1%Pen/Strep
  4. 70%乙醇
    将700ml 100%乙醇与300ml去离子水混合
  5. 碱性成纤维细胞生长因子(bFGF)
    在1ml含有0.1%BSA的5mM Tris(pH7.6)中稀释50μgbFGF(50μg/ml)。
  6. 鸡胚提取物(CEE)
    在含有冻干CEE(50μg/ml)的瓶中加入10ml PBS
  7. 肌纤维生长培养基(MfGM)
    补充含有10%FBS,1%Pen/Strep,2%CEE和10ng/ml bFGF的DMEM。 在37℃下在水浴中温育MfGM 使用前,用0.22μm过滤器过滤MfGM 注意:对于两个EDL肌肉,准备36ml的MfGM。
  8. 4%多聚甲醛(PFA)
    将0.4g PFA在10ml PBS中稀释 在60℃和250rpm下剧烈振荡1-2小时,直至完全溶解 使用前,用0.45μm过滤器过滤4%PFA
  9. 10%Triton X-100 将100μl100%Triton X-100与900μlPBS混合
  10. 淬火溶液
    对于10ml溶液,将75.07mg甘氨酸与200μl10%Triton X-100和50μl5%叠氮化钠组合。
    用PBS完成10ml。
  11. 封锁解决方案
    对于10ml溶液,将200mg BSA与200μl10%Triton X-100和500μl马血清合并。
    用PBS完成10ml。
    使用前,用0.45μm过滤器过滤阻塞溶液。

致谢

这项工作得到了来自NIH授权AR059810,AR068313和AG029623(到A.Kumar)和AR069985(到S.M.P Hindi)的资助。

参考文献

  1. Anderson,JE,Wozniak,AC and Mizunoya,W.(2012)。  用于细胞,分子,药理学和进化研究的单一肌纤维分离和培养。 方法Mol Biol 798:85-102。
  2. Bischoff,R。(1986)。  肌肉卫星的增殖细胞在培养物中的完整肌纤维上。 Dev Biol 115(1):129-139。
  3. 印度语,SM,Paul,PK,Dahiya,S.,Mishra,V.,Bhatnagar,S.,Kuang,S.,Choi,Y.和Kumar,A。(2012)。  TRAF6和缺刻信号之间的相互作用调节成年肌纤维再生损伤。 Mol Cell Biol 32(23):4833-4845。
  4. Hindi,SM和Kumar,A.(2016)。  TRAF6调节卫星干细胞自我更新和再生性肌发生期间的功能。 126(1):151-168。
  5. Kuang,S.,Charge,SB,Seale,P.,Huh,M.and Rudnicki,MA(2006)。 
  6. Kuang,S. and Rudnicki,MA(2008)。  卫星细胞的新兴生物学及其治疗潜力。趋势Mol Med 14(2):82-91。
  7. Keire,P.,Shearer,A.,Shefer,G.and Yablonka-Reuveni,Z.(2013)。  分离和培养骨骼肌肌纤维作为分析卫星细胞的方法。 Methods Mol Biol 946:431-468。
  8. Le Grand,F。和Rudnicki,MA(2007)。  Skeletal muscle satellite cells and adult myogenesis。Curr Opin Cell Biol 19(6):628-633。
  9. Ogura,Y.,Hindi,SM,Sato,S.,Xiong,G.,Akira,S. and Kumar,A。(2015)。  TAK1调节卫星干细胞内环境稳定和骨骼肌修复。 Nat Commun 6:10123.
  10. Ogura,Y.,Mishra,V.,Hindi,SM,Kuang,S.和Kumar,A.(2013)。  从成体骨骼肌分离和培养个体肌纤维和它们的卫星细胞。(73):e50074。
  11. Rosenblatt,JD,Lunt,AI,Parry,DJ和Partridge,TA(1995)。  从活单肌纤维外植体培养卫星细胞。 In Vitro Cell Dev Biol Anim 31(10):773-779。
  12. Shefer,G.和Yablonka-Reuveni,Z。(2005)。  骨骼肌肌纤维的分离和培养作为分析卫星细胞的手段。

    Methods Mol Biol 290:281-304。
  13. Siegel,AL,Atchison,K.,Fisher,KE,Davis,GE和Cornelison,DD(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 gov/pubmed/19609936"target ="_ blank">肌肉卫星细胞运动性的3D时间间隔分析。干细胞 27(10):2527-2538。
  14. Verma,M.和Asakura,A。(2011)。  用于卫星细胞检测的β-半乳糖苷酶染色后,从酒精固定的成体肌肉中分离有效的单一肌肉纤维。 59 Histochem Cytochem 59(1):60-67。

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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gallot, Y. S., Hindi, S. M., Mann, A. K. and Kumar, A. (2016). Isolation, Culture, and Staining of Single Myofibers. Bio-protocol 6(19): e1942. DOI: 10.21769/BioProtoc.1942.
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