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Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice
成年小鼠骨骼肌的原代成肌细胞分离、培养和分化   

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Abstract

Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been suggested as the most physiologically relevant model for studying myogenesis in vitro. However, due to their low abundance in adult skeletal muscle, isolation of primary myoblasts is technically challenging. In this article, we describe an improved protocol for the isolation of primary myoblasts from adult skeletal muscle of mice. We also describe methods for their culturing and differentiation into myotubes.

Keywords: Myoblast(成肌细胞), Skeletal muscle(骨骼肌), Myogenesis(肌细胞生成), MyoD(MyoD), Pax7(Pax7), Myogenin(肌细胞生成素), Myogenic differentiation(成肌分化), Hind limb muscle(后肢肌肉)

Background

Myogenesis is a complex and highly orchestrated process that involves the determination of multipotential mesodermal cells to give rise to myoblasts, exit of myoblasts from the cell cycle, and their eventual differentiation into skeletal muscle fibers. Myogenesis is regulated by the sequential expression of myogenic regulatory factors (MRFs), a group of basic helix-loop-helix transcription factors that include Myf-5, MyoD, myogenin, and MRF4. Myf-5 and MyoD are the primary MRFs required for the formation, proliferation, and survival of myoblasts, whereas other MRFs such as myogenin and MRF-4 act late during myogenesis, activating gene expression of contractile proteins and other structural and metabolic proteins (Buckingham et al., 2003; Bentzinger et al., 2012).

Myogenesis is also regulated by a number of transcription factors and several noncoding RNAs, which act at specific steps including commitment of progenitor (satellite) cells to myogenic lineage and myoblast proliferation, differentiation, and fusion (Yin et al., 2013; Simionescu-Bankston and Kumar, 2016). Initial experiments for studying the role of various regulatory proteins in myogenesis are performed using cultured myoblasts. There are several myoblastic cell lines (e.g., C2C12, L6, BC3H1, and MM14) that differentiate into myotubes upon incubation in differentiation medium. These cell lines have also been used to establish myotube cultures to investigate the effects of various molecules on myotube growth and atrophy. However, there is often some degree of variability in results potentially due to the origin of cells, culture conditions, and passage number. The use of primary myoblasts is highly recommended because they are devoid of the side-effects characteristic of the immortalization process and their physiological relevance to the living organisms. Primary myoblasts can be isolated from the skeletal muscle of neonatal or adult mice. However, the process of isolation of myoblasts from neonatal muscle is more complex because it also requires Percoll density gradient centrifugation (Dogra et al., 2006). Some investigators also use fluorescence-activated cell sorting (FACS) approach to isolate primary myoblasts from digested muscle tissues especially to study regulation of quiescence and activation of these cells. However, FACS sorting is an expensive approach which requires several negative and positive selection antibodies and a cell sorter machine. Moreover, the yield of myoblasts is generally low and there are always chances of contamination during isolation of purified myoblasts by FACS technique. In our laboratory, we have adapted and standardized a previously published protocol (Rando and Blau, 1994) for the isolation of myoblasts from skeletal muscle of adult mice. This protocol is highly efficient for the generation of a large amount of purified myoblasts from skeletal muscle of adult mice (Ogura et al., 2015; Hindi and Kumar, 2016). The purity of the myoblasts can be assayed by immunostaining of the cells for Pax7 and MyoD proteins which are expressed in undifferentiated myoblasts. Moreover, primary myoblasts isolated using this protocol efficiently differentiate into multinucleated myotubes on incubation in differentiation medium and myotubes can be readily visualized by phase contrast microscopy or after immunostaining for myosin heavy chain (MyHC), a protein expressed in differentiated muscle cells (Hindi et al., 2014; Bohnert et al., 2016). Finally, like myogenic cell lines, the purified primary myoblasts can be stored in liquid nitrogen or -80 °C for unlimited time and can be regrown whenever required.

Materials and Reagents

  1. Sterilization pouches (Fisher Scientific, catalog number: 01-812-51 )
  2. 100 x 20 mm-Petri dishes (Corning, catalog number: 430167 )
  3. 6-well plates (Corning, Falcon®, catalog number: 353046 )
  4. 1.5 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5510 )
  5. 0.22 μm filter (EMD Millipore, catalog number: SLGP033RS )
  6. 15 ml sterile tubes (VWR, catalog number: 89004-368 )
  7. 1 ml pipette tip
  8. Parafilm
  9. 10 ml serological pipette (Santa Cruz Biotechnology, catalog number: sc-200281 )
  10. 70 µm strainer (Fisher Scientific, catalog number: 22-363-548 )
  11. 50 ml sterile tubes (VWR, catalog number: 89004-364 )
  12. 30 µm filters (Milteny Biotech, catalog number: 130-041-407 )
  13. 0.45 μm filter (EMD Millipore, catalog number: SLHV033RS )
  14. 24-well plates (Corning, Falcon®, catalog number: 353047 )
  15. Slip-tip syringe (BD, catalog number: 302833 )
  16. Sterile cell scraper (Corning, Falcon®, catalog number: 353085 )
  17. Adult mice (Mus musculus; 6-8-weeks old) (see Notes 7 and 8)
  18. 2,2,2-tribromoethanol (Avertin) (Sigma-Aldrich, catalog number: T48402 )
  19. 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  20. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  21. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  22. Primary antibody anti-Pax7 (mouse) (Developmental Studies Hybridoma Bank, catalog number: Pax7 )
  23. Primary antibody anti-MyoD (rabbit) (Santa Cruz Biotechnology, catalog number: sc-304 )
  24. Primary antibody anti-MyHC (mouse) (Developmental Studies Hybridoma Bank, catalog number: MF-20 )
  25. Secondary antibody goat anti-rabbit Alexa Fluor® 488 conjugate (Thermo Fisher Scientific, Invitrogen, catalog number: A-11034 )
  26. Secondary antibody goat anti-mouse Alexa Fluor® 568 conjugate (Thermo Fisher Scientific, Invitrogen, catalog number: A-11004 )
  27. 100% ethanol (Decon Labs, catalog number: 2701 )
  28. Matrigel (Corning, catalog number: 354234 )
  29. Dulbecco’s modified Eagle’s medium (DMEM) high glucose, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11995065 )
  30. Collagenase II (Worthington Biochemical, catalog number: LS004176 )
  31. Ultra-pureTM water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  32. Penicillin-streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  33. N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  34. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
  35. Recombinant human fibroblast growth factor-basic (bFGF) (PeproTech, catalog number: 100-18B )
  36. Tris base (Fisher Scientific, catalog number: BP152-5 )
  37. F-10 Nutrient mixture (Thermo Fisher Scientific, GibcoTM, catalog number: 11550043 )
  38. Dulbecco’s modified Eagle’s medium (DMEM) (ATCC, catalog number: 30-2002 )
  39. Horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26050088 )
  40. Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-100 )
  41. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  42. 100% Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
  43. 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, catalog number: D8417 )
  44. 70% ethanol (see Recipes)
  45. 10% Matrigel (see Recipes)
  46. Collagenase II (see Recipes)
  47. Digestion medium (see Recipes)
  48. Collection/washing/mincing solution (see Recipes)
  49. Neutralization/isolation media (see Recipes)
  50. Basic fibroblast growth factor (bFGF) (see Recipes)
  51. Myoblast growth medium (MGM) (see Recipes)
  52. Post isolation washing medium (see Recipes)
  53. Differentiation medium (DM) (see Recipes)
  54. Freezing medium (see Recipes)
  55. 4% paraformaldehyde (PFA) (see Recipes)
  56. 0.3% Triton X-100 (see Recipes)
  57. 10% Triton X-100 (see Recipes)
  58. Blocking solution (see Recipes)
  59. DAPI (see Recipes)

Equipment

  1. Autoclave
  2. Dissection tools: Sterilized/autoclaved scissors and forceps
  3. Biosafety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series Class II , Type A2)
  4. Bench top centrifuge for 1.5 ml Eppendorf tubes (Eppendorf, model: 5424/5424 R )
  5. Bench top centrifuge for 15 ml and 50 ml tubes (Eppendorf, model: 5702/5702 R/5702 RH )
  6. Heated incubator shaker (Eppendorf, New BrunswickTM, model: Excella E24 )
  7. CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3578 )
  8. Microscope (Nikon Instruments, model: Eclipse TE2000 )
  9. Water bath (Thermo Fisher Scientific, model: Model 215 , catalog number: 15-462-15Q)
  10. 500 ml bottle

Procedure

The basic steps for isolation and purification of primary myoblasts from hind limb muscle of mice are presented in Figure 1.


Figure 1. The schematic view of general procedures for the isolation of primary myoblasts from hind limb muscle of mice. MGM, myoblast growth medium; FGF, fibroblast growth factor; DM, differentiation medium.

  1. Before isolation of primary myoblasts
    1. Autoclave two sets of tools in sterilization pouches each containing one small pair of scissors and forceps (see Notes 1 and 2).
    2. Following Recipe 1, prepare 70% ethanol.
    3. Following Recipe 2, prepare 10% Matrigel (see Note 3).
    4. Following Recipe 3, prepare collagenase II (see Note 4).
    5. Following Recipe 4, prepare base of digestion medium (see Notes 5 and 6).
    6. Following Recipe 5, prepare collection/washing/mincing solution.
    7. Following Recipe 6, prepare neutralization/isolation medium (see Note 5).
    8. Following Recipe 8, prepare myoblast growth medium (MGM) (see Note 5).
    9. Coat 100 x 20 mm Petri dishes (one plate per mouse) by pouring and spreading 10% Matrigel solution enough to cover the plate area. Avoid bubbles. After 30 sec, remove excess Matrigel solution (excess 10% Matrigel can be collected and stored for re-use multiple times, if kept sterile). Leave the plate without cap to dry in sterile air flow in a biosafety cabinet (~20 min). Once dried, put lid on the plate and leave in sterile biosafety cabinet at room temperature until ready to use. There is no need to turn on UV light of biosafety cabinet. This plate will be used for isolation and culturing of myoblasts following digestion of muscle with collagenase.
    10. For muscle collection and washing before digestion, prepare a 6-well plate containing 3 ml/well of collection/washing/mincing solution (only 4 wells of a 6-well plate needed per mouse).
    11. For muscle mincing following isolation, prepare two 1.5 ml Eppendorf tubes per mouse each containing 500 µl of collection/washing/mincing solution.

  2. Muscle removal and washing
    1. Euthanize mouse following approved IACUC guidelines in your laboratory. For purpose of this protocol, the mouse was given intraperitoneal injection of 2,2,2-tribromoethanol at a dose of 250 mg/kg, followed by cervical dislocation.
    2. The isolation of hind limb muscles from mice is performed outside a biosafety cabinet. For this step, no sterile techniques are necessary except autoclaved dissection tools and spray of ethanol. Spray hind-limbs with 70% ethanol and pin the mouse face up.
    3. Remove skin using autoclaved scissors from both hind-limbs to expose muscles.
    4. Using the first pair of autoclaved scissors and forceps, collect tibial anterior, gastrocnemius, soleus, quadriceps and extensor digitorum longus muscles from one hind-limb and place them into one well of 6-well plate containing collection/washing/mincing solution (Video 1).

      Video 1. Isolation of hind limb muscles from mouse. This video shows basic steps for isolation of hind limb muscles from euthanized mouse and transferring them into a 6-well plate containing collection/washing/mincing solution.

    5. Repeat the previous step on the other hind limb and place the muscles in the second well of 6-well plate containing collection/washing/mincing solution (see Note 9).
    6. Transfer the muscles using the second pair of autoclaved forceps to the other two wells of the 6-well plate filled with collection/washing/mincing solution. This is needed to wash and remove any sticking hair or unwanted tissue on isolated muscles.
    7. Put the lid on the 6-well plate containing muscle and collection/washing/mincing solution and spray with 70% ethanol before transferring the plate to a sterile biosafety cabinet. From this step onwards, all the procedure must be performed in biosafety cabinet to avoid potential contamination of myofibers. 

  3. Muscle mincing/digestion
    1. Transfer washed muscles using second pair of autoclaved forceps to two 1.5 ml Eppendorf tubes (one tube for each hind limb muscle group) containing collection/washing/mincing solution and mince muscles very finely using second pair of autoclaved scissors (see Note 10).
    2. Once muscles are minced, spin-down tubes using an RT centrifuge at 21,130 x g for 30 sec to separate the minced muscle pieces from the collection/washing/mincing solution.
    3. Set spun tubes aside under sterile biosafety cabinet.
    4. Complete preparation of digestion media by adding 1 ml of collagenase II to base of digestion media prepared previously (see Note 6). Filter the entire digestion mixture using a 0.22 μm syringe filter into a 15 ml tube.
    5. Remove the supernatant from the Eppendorf tubes containing the minced muscles (in step C2).
    6. To transfer the minced muscles to digestion media tube, cut off the tip of a sterile 1 ml pipette tip to create a large bore to accommodate minced muscles’ size.
    7. Transfer a small amount of digestion media to the Eppendorf tubes to resuspend minced muscle and then transfer the mix (minced muscles + digestion media) to the original 15 ml digestion media tube (in step C4).
    8. Repeat step C7 until all the minced muscles are transferred from the Eppendorf tubes to the 15 ml tube containing digestion mixture.
    9. Close the lid of the 15 ml tube containing minced muscle and digestion media and wrap the lid with Parafilm to avoid any potential leakage.
    10. Vortex tube for 5 sec.
    11. Place tube horizontally in a 37 °C shaker and shake at 100 rpm for 1 h with another 5 sec vortex half way through (see Note 11).
    12. After 1 h, mix should appear cloudy and muscle pieces should be mostly digested and very small (see Note 12).
    13. Once digestion is complete, vortex tube containing digested muscle mixture for 5 sec.
    14. Spin the tubes at 1,400 x g for 5 min at room temperature.
    15. Under sterile biosafety cabinet, remove supernatant (digestion media) and discard.
    16. Resuspend digested muscle pellet in 7 ml of neutralizing/isolation media using a 10 ml serological pipette. Pipet the digested muscle tissue pellet up and down for 20-30 times using a sterile 10 ml pipette. This ensures that most myoblasts are released from the muscle tissues.
    17. Place a 70 μm strainer on a 50 ml tube.
    18. Pre-wet the 70 μm strainer with 2 ml of neutralizing/isolation media.
    19. Collect resuspended muscle pellet mixture (step C16) using a 10 ml serological pipette and pass through the pre-wet 70 μm strainer.
    20. Wash the strainer with 2 ml of neutralizing/isolation media to ensure that cells are sticking to strainer (most tissue debris will be removed during this step).
    21. In a 15 ml tube, pre-wet a 30 μm filter with 1 ml of neutralizing/isolation media.
    22. Pass cell mixture (previously strained through 70 μm strainer) through the pre-wet 30 μm filter. This step will remove large infiltrating cells such as macrophages from the myoblasts.
    23. Wash filter with 1 ml of neutralizing/isolation media to ensure that cells are not sticking to the strainer.
    24. Spin tubes containing cellular mixture at 1,400 x g for 5 min.
    25. Remove supernatant (neutralizing/isolation media) and resuspend cell pellet in 10 ml MGM + bFGF.
    26. Seed the cellular mixtures onto the 10% Matrigel pre-coated dish.
    27. Visualize the cells under a microscope. At this step, there will be a heterogeneous mixture of muscle particles and cells (Figure 2).


      Figure 2. Representative phase contrast microscopy images of primary myoblast cultures at various stages of purification. Digested muscle mixture following filtration at seeding in 10% Matrigel-coated culturing dish at (A) 0 h, (B) 24 h, and (C) 72 h. Appearance of myoblasts after (D) first pre-plating and (E) second pre-plating steps. F. Purified myoblast cultures after 2-4 pre-plating steps. Scale bars = 20 µm. Blue arrows point to tissue/cellular debris; Red arrows point to fibroblasts (large and flat triangular-shaped) and green arrows point to myoblasts (round or bipolar spindle-shaped).

    28. Keep the dish in a 37 °C CO2 incubator for 72 h. Do not change medium during this incubation period.
    29. After 72 h, you should be able to visualize adherent, small, droplet-shaped myoblasts and large triangular-shaped fibroblasts in addition to remnant tissue debris.
    30. At this time, the cells are ready for first pre-plating (see Note 13).

  4. Myoblast purification (Pre-Plating)
    1. Remove culture media from the dish.
    2. Using a 10 ml serological pipette, gently wash adherent cells two times with post-isolation media (F-10 medium containing 1% Pen/Strep).
    3. Detach cells by adding 2 ml of 0.25% trypsin-EDTA and placing cells at 37 °C in a CO2 incubator for 2 min.
    4. Visualize under the microscope to ensure that all cells are detached.
    5. Once cells are detached, add 10 ml of MGM + bFGF to dish (see Note 14).
    6. Transfer cell mixture to a non-Matrigel coated tissue culture dish and place at 37 °C in a CO2 incubator for 45 min. This will allow larger cells such as fibroblasts to adhere in the bottom of the dish while most myoblasts would still be suspended in the media.
    7. During this time, coat a new Petri dish with 10% Matrigel as described above.
    8. After 45 min, transfer all supernatant from pre-plating dish to the new Matrigel-coated dish and place the dish in CO2 incubator (see Note 15).
    9. Pre-plating step should be performed every 36-48 h until > 98% myoblast purity is achieved (see Note 16). Representative phase contrast images at various steps of myoblast isolation are presented in Figures 2C-2E. Purified myoblast culture image is presented in Figure 2F.

  5. Culturing of myoblasts
    1. The purified myoblasts are maintained in culture by incubation in the myoblast growth medium and changing media every other day.
    2. Do not allow cells to reach > 70% confluency (unless experimentally required) as this will lead to pre-mature differentiation and fusion of myoblasts with each other.
    3. Expand cells by splitting into multiple 10% Matrigel-coated Petri dishes as needed.
    4. The myoblasts can be collected in freezing medium and stored in liquid nitrogen tank or -80 °C freezer like any other cultured cells. Primary myoblasts maintain their proliferation and differentiation capacity once thawed and cultured again.

  6. Differentiating primary myoblasts to myotubes
    1. Coat tissue culture plates with 10% Matrigel similar to those described above.
    2. Plate primary myoblasts in pre-coated Matrigel plates. For obtaining good quality myotubes, myoblasts should be at 85-95% confluency before adding differentiation medium (DM). For a 6-well tissue culture plate, we need 0.75-0.9 x 106 cells per well; however, this number will vary depending on the cell passage number. Low passage cells are normally smaller in size therefore use the higher end of the range to achieve desired confluency, whereas higher passage cells are more elongated and larger in size so the lower end of cell number will be required (see Note 17).
    3. Next day, replace myoblast growth medium with DM (see Note 18).
    4. DM should be changed every 48 h.
    5. Myoblasts align and fusion starts within 24-36 h after incubation in DM. At 48 h, small myotubes can be clearly seen, and by 72-96 h they become large and mature.

  7. Immunostaining of primary myoblasts and myotubes
    1. Remove culture media from 6-well tissue culture plates containing primary myoblasts or myotubes which are adhered to surface of the wells.
    2. Fix adhered cells by adding appropriate amount of 4% PFA to cover cells and incubate 15 min.
    3. Remove 4% PFA and gently wash the cells three times with 1x PBS for 5 min each.
    4. Permeabilize the cells by adding 0.3% Triton X-100 and incubate for 7 min at room temperature.
    5. Remove 0.3% Triton X-100 and gently wash cells three times with PBS for 5 min each.
    6. Remove PBS and block cells by adding 2% BSA solution for 20 min.
    7. Prepare primary antibody mixtures in blocking solution.
      Note: We use 1:10 dilution of Pax7 antibody, 1:200 dilution of MyoD, and 1:200 dilution of MF-20 (i.e., MyHC) antibody.
    8. Remove blocking solution and add desired primary antibody mixture to each well.
    9. Wrap plates with Parafilm to prevent evaporation of primary antibody solution.
    10. Primary antibody incubation can be performed for 1-2 h at room temperature or overnight at 4 °C.
    11. Once primary antibody incubation period is completed, remove primary antibody solution and wash cells three times with PBS for 5 min each.
    12. Prepare secondary antibody mixture in blocking solution at a concentration of 1:1,500 for each secondary antibody.
      Note: We use anti-mouse Alexa 568 for red fluorescence and anti-rabbit Alexa 488 for green fluorescence.
    13. Wrap plates with Parafilm to prevent evaporation of secondary antibody.
    14. Secondary antibody incubation is performed at room temperature for 1 h in the dark.
    15. Remove secondary antibody solution and wash cells three times with PBS for 5 min each.
    16. Prepare DAPI working solution in PBS.
    17. Remove PBS from the wells and add DAPI solution for staining nuclei.
    18. DAPI incubation is performed at room temperature for 3 min in the dark.
    19. Remove DAPI and wash the cells three times with PBS for 5 min each.
    20. Add PBS to the wells.
    21. The cells are now ready to be visualized under a fluorescent microscope. Representative images of myoblast culture after staining with anti-Pax7, anti-MyoD, and DAPI are presented in Figure 3. Differentiated myotube culture images after staining with anti-MyHC and DAPI are presented in Figure 4.


      Figure 3. Staining of primary myoblast culture for Pax7 and MyoD. Primary myoblast cultures grown in MGM were fixed and immunostained for Pax7 and MyoD protein. Nuclei were counterstained with DAPI. Representative individual anti-Pax7, anti-MyoD, and DAPI-stained and merged images (triple stained) are presented here. Scale bars = 20 µm.


      Figure 4. Staining of primary myotube cultures for myosin heavy chain (MyHC). Primary myoblasts were incubated in DM for 24 h or 48 h. The cultures were fixed and immunostained for MyHC protein. Nuclei were counterstained with DAPI. Representative individual anti-MyHC and DAPI-stained and merged images are presented here. Scale bars = 20 µm.

Data analysis

For individual experiments, the sample size should be determined by power analysis. For most of our studies focused on studying the effect of regulatory factors on myoblast proliferation and differentiation, we perform experiments in 4-5 replicates. For studying myogenic differentiation, we calculate myogenic index which is percentage of nuclei in MyHC-stained myotubes in total nuclei in the plate. We present the data as mean ± standard deviation (SD). We use paired or unpaired Student’s t-test to determine statistical differences among different groups similar to as described (Ogura et al., 2015; Hindi and Kumar, 2016). A P < 0.05 is considered as statistically significant.

Notes

  1. All steps except initial isolation of hind limb muscle should be performed under sterile conditions. Even for the isolation of hind limb muscle from euthanized mice, use of autoclaved dissection tools and spray ethanol is highly recommended.
  2. One set will be used to collect muscle from mouse while the other set will be used under the sterile biosafety cabinet to transfer and mince muscle.
  3. Do not allow 100% Matrigel to reach RT as this will cause it to solidify and form a thick gel like material that would be difficult to dilute in DMEM.
  4. Worthington’s powder stock of collagenase does not have same activity (i.e., units per mg dry weight) in all the batches. Therefore, carefully read the activity present in the stock powder vial when preparing collagenase stock each time.
  5. All media should be warmed at 37 °C before use.
  6. Collagenase should only be added when muscle is minced and ready to be digested as adding collagenase ahead of time will weaken its digestive activity.
  7. To obtain primary myoblasts from adult satellite cells mice must be ≥ 6 weeks of age.
  8. To obtain optimal primary myoblasts mice should not be ≥ 8 weeks of age. This protocol can be performed on younger or older mice if experimental settings require so however some alterations in myogenic yield and purity may be observed; younger mice will yield more myoblasts as their muscle contain actively proliferating cells that are contributing to muscle development and growth. On other hand, older or diseased mice may yield less myoblasts with lower purity and higher fibroblast content. Similar consideration should be taken into account when isolating primary myoblasts from genetic mouse models or mice subjected to various experimental procedures.
  9. Muscle collection from both limbs should be performed within 5 min to preserve viability.
  10. Make sure that muscles are finely minced at this step as this will affect the yield of myoblasts.
  11. It is important to observe the appearance of the digesting muscle mixture at this point. Muscle pieces should look smaller and the digestion medium should start to appear cloudy. If muscle pieces are very small then reduce the second half of the digestion time to 20 min.
  12. If muscle pieces still appear larger in size and do not seem to be completely digested, extend the digestion time for another 10 min or until digestion appears to be complete.
  13. If cell density is very low 72 h post isolation, do not pre-plate at this time, just wash dish with F-10 + 1% PS and re-feed with fresh MGM + bFGF.
  14. bFGF is either added directly to the culturing dish at a concentration of 10 ng/ml, or a mixture of MGM + bFGF can be prepared ahead of time at a similar concentration, however this is only good for 1-week if stored properly at 4 °C.
  15. Sometimes myoblasts also settle to the bottom of the pre-plating dish, for that reason add media to that dish as well then next day if a large amount of myoblasts is observed repeat the pre-plating step for a shorter duration (~20 min).
  16. Although high myoblast purity can usually be achieved by performing 1-4 pre-plating repetitions, under certain circumstances this does not suffice. If myoblasts do not seem to be increasingly dominating the culture with each pre-plating step then one can resolve to an alternative purification strategy:
    1. Split your cultures into multiple Petri dishes so that each dish contains a cellular mixture that is around 10% confluent.
    2. Each cell should appear spatially separated from its neighboring cells.
    3. To allow cells to grow continuously change MGM every other day.
    4. With each day you should observe the formation of myoblast colonies originating from a single cell. You will also observe the formation of fibroblast colonies originating from a single cell.
    5. Under an inverted microscope, locate the areas where fibroblast colonies are observed and label those areas by marking the bottom of the dish using a marker.
    6. Under the sterile biosafety cabinet, physically scrape off the fibroblast colonies (marked in Note 16e) using a sterile cell scraper.
    7. Aspirate the culturing medium and wash your dish a couple of times with post-isolation washing medium.
    8. Trypsinize the remaining cells (should be pre-dominantly myoblast colonies) left over after scraping and washing off fibroblast colonies in Notes 16f and 16g.
    9. Plate your cells in a newly coated 10% Matrigel dish.
    10. High myoblast purity should be observed.
  17. Avoid overcrowding the well with cells when plating for differentiation, as this will restrict the space that is required for myoblasts to elongate prior to fusion and impose extra stress on the cells, possibly leading to cell death and defective myotube formation.
  18. If cells are not at optimum confluency next day, allow cells to proliferate longer to achieve desired confluency before switching to DM.

Recipes

  1. 70% ethanol
    Combine 700 ml of 100% ethanol with 300 ml of deionized water
  2. 10% Matrigel
    1. Thaw Matrigel in ice overnight
    2. Dissolve 10 ml cold Matrigel in 90 ml of cold DMEM
    3. Aliquot 5 ml of 10% Matrigel per 15 ml sterile tube into multiple tubes for later use
    4. Store 10% Matrigel aliquots at -20 °C for long term storage
    5. For frequent use, store 10% Matrigel tube at 4 °C
    Note: Do not re-freeze 10% Matrigel solution once removed from -20 °C and thawed.
  3. Collagenase II
    1. Prepare stock by dissolving collagenase II in ultra-pure water at a concentration of 4,000 U/ml
    2. Distribute this collagenase II stock solution into multiple 1 ml aliquot in sterile Eppendorf tubes for later use
    3. Store aliquots at -20 °C
    4. Thaw collagenase II immediately before use
    Note: Avoid repetitive freeze-thaw cycles as this would reduce the enzymatic activity of collagenase II.
  4. Digestion medium
    10 ml digestion media is enough for digesting hind limb muscle of one mouse, volume can be modified if older mice are used
    For 10 ml:
    1. Prepare base of digestion medium, by supplementing 8.65 ml DMEM (Gibco) with 100 μl of Pen/Strep (1%) and 250 μl of HEPES (2.5%). Keep at 37 °C in a water bath until ready for use
    2. Once muscle has been isolated, minced and ready for digestion, add collagenase II to make a final concentration of 400 U/ml. Shake briefly by hand
    3. Filter digestion medium using a 0.22 μm filter
  5. Collection/washing/mincing solution
    For 15 ml, combine 150 μl of Pen/Strep with 14.85 ml of 1x PBS to achieve a solution of 1% Pen/Strep in PBS
  6. Neutralizing/isolation medium
    To prepare a 500 ml stock of neutralizing medium:
    1. Remove 50 ml of DMEM (Gibco) from 500 ml bottle and store at 4 °C for any later uses
    2. Add 50 ml of filtered FBS to DMEM bottle now containing 450 ml DMEM to achieve a concentration of 10% FBS
    3. Add 5 ml of Pen/Strep to above mix to achieve a concentration of 1% Pen/Strep
    4. Store this stock at 4 °C
    5. Warm desired amount at 37 °C before use
  7. Basic fibroblast growth factor (bFGF)
    1. Dilute 50 μg of bFGF (50 μg/ml) in 1 ml of filter-sterilized 5 mM Tris (pH 7.6) with 0.1% BSA
    2. Aliquot into small (50-100 μl) and store at -20 °C or -80 °C
    3. Thaw aliquot immediately before use
    Note: Avoid repetitive freeze-thaw cycles.
  8. Myoblast growth medium (MGM)
    To prepare a 500 ml stock of MGM:
    1. Remove 100 ml of F-10 media from a 500 ml bottle and store the removed media at 4 °C for any potential use
    2. Add 100 ml of filtered FBS to 400 ml F-10 medium. Add 5 ml of Pen/Strep solution
    3. Store the media at 4 °C
    4. Warm desired amount of this media at 37 °C before use
    5. Add 10 ng/ml basic fibroblast growth factor (bFGF) just before using the media for cells (Note 12)
    6. Each dish will require 10 ml of MGM
  9. Post-isolation washing medium
    For 50 ml:
    Supplement 49.5 ml of F-10 media with 500 μl of Pen/Strep to achieve a concentration of 1% Pen/Strep
    This can be stored at 4 °C. Warm at 37 °C before use
  10. Differentiation medium (DM)
    For 500 ml stock:
    1. Remove 15 ml of DMEM (ATCC) from a 500 ml bottle
    2. Add 10 ml of filtered horse serum to the DMEM bottle
    3. Add 5 ml of Pen/Strep to above mixture to achieve a concentration of 1% Pen/Strep
    4. Store this stock at 4 °C
    5. Warm desired amount at 37 °C before use
  11. Freezing medium
    For 10 ml:
    Combine 5 ml of MGM (see Recipe 8) with 4 ml of filtered FBS (40% FBS) and 1 ml of DMSO (10%)
    This can be stored at 4 °C and warmed just before use
  12. 4% paraformaldehyde (PFA)
    For 10 ml:
    1. Suspend 0.4 g of PFA in 10 ml of PBS
    2. Shake at 250 rpm at 60 °C for 1-2 h until completely dissolved
    3. Allow to cool at room temperature and filter by passing through a 0.45 μm syringe filter
    4. Store at 4 °C
    5. For optimal results, do not use 4% PFA if stored more than 2 days
  13. 0.3% Triton X-100
    1. Prepare a stock of 10% Triton X-100 by combining 1 ml of 100% Triton X-100 with 9 ml of PBS
    2. To prepare 10 ml of 0.3% Triton X-100, add 300 μl of 10% Triton X-100 to 9.7 ml PBS
  14. Blocking solution
    For 50 ml:
    1. Dissolve 1 g BSA in 50 ml of PBS
    2. Filter using a 0.45 μm syringe filter
    3. This can be stored at -20 °C for future use
  15. DAPI
    For 10 ml:
    Add 2 μl of DAPI stock solution to 10 ml of PBS and filter using a 0.45 μm syringe filter
    The working solution of DAPI can be stored in the dark at 4 °C

Acknowledgments

This work was supported by funding from NIH grants AR059810, AR068313, and AG029623 (to A. Kumar) and AR069985 (to S. M. Hindi). This protocol has been adapted and slightly modified from previously published articles (Rando and Blau, 1994; Hindi et al., 2014; Ogura et al., 2015; Hindi and Kumar, 2016).

References

  1. Bentzinger, C. F., Wang, Y. X. and Rudnicki, M. A. (2012). Building muscle: molecular regulation of myogenesis. Cold Spring Harb Perspect Biol 4(2).
  2. Bohnert, K. R., Gallot, Y. S., Sato, S., Xiong, G., Hindi, S. M. and Kumar, A. (2016). Inhibition of ER stress and unfolding protein response pathways causes skeletal muscle wasting during cancer cachexia. FASEB J 30(9): 3053-3068.
  3. Buckingham, M., Bajard, L., Chang, T., Daubas, P., Hadchouel, J., Meilhac, S., Montarras, D., Rocancourt, D. and Relaix, F. (2003). The formation of skeletal muscle: from somite to limb. J Anat 202(1): 59-68.
  4. Dogra, C., Changotra, H., Mohan, S. and Kumar, A. (2006). Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein. J Biol Chem 281(15): 10327-10336.
  5. 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.
  6. Hindi, S. M., Mishra, V., Bhatnagar, S., Tajrishi, M. M., Ogura, Y., Yan, Z., Burkly, L. C., Zheng, T. S. and Kumar, A. (2014). Regulatory circuitry of TWEAK-Fn14 system and PGC-1alpha in skeletal muscle atrophy program. FASEB J 28(3): 1398-1411.
  7. 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.
  8. Rando, T. A. and Blau, H. M. (1994). Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125(6): 1275-1287.
  9. Simionescu-Bankston, A. and Kumar, A. (2016). Noncoding RNAs in the regulation of skeletal muscle biology in health and disease. J Mol Med (Berl) 94(8): 853-866.
  10. Yin, H., Price, F. and Rudnicki, M. A. (2013). Satellite cells and the muscle stem cell niche. Physiol Rev 93(1): 23-67.

简介

造血是一种多步骤过程,导致在损伤的肌纤维的胚胎发育和修复期间骨骼肌的形成。在这个过程中,成肌细胞是主要的效应细胞类型,彼此融合或损伤肌纤维,导致新成肌纤维的形成或成年人骨骼肌的再生。通过体外成骨细胞分化成肌管可以概括出许多发生肌肉发育的步骤。大多数实验室使用也分化成肌管的永生化肌原细胞系。虽然已经发现这些细胞系对于描绘造血的调节机制非常有用,但是它们通常依赖于细胞的来源和培养条件而显示出很大的变异性。原代成肌细胞被认为是体外研究肌生成的最生理学相关模型。然而,由于成体骨骼肌的丰度低,原代成肌细胞的分离在技术上是有挑战性的。在本文中,我们描述了一种用于从小鼠的成年骨骼肌分离原代成肌细胞的改进方案。我们还描述了其培养和分化成肌管的方法。


背景 造血是一个复杂而高度协调的过程,其涉及多潜能中胚层细胞的测定,以产生成肌细胞,成肌细胞从细胞周期中排出,以及它们最终分化为骨骼肌纤维。 Myogen-5,MyoD,myogenin和MRF4的基因螺旋 - 环 - 螺旋转录因子的一组基因调控因子(MRFs)的顺序表达调控。 Myf-5和MyoD是成肌细胞形成,增殖和存活所需的主要MRFs,而其他MRF(如肌细胞生成素和MRF-4)在肌发生过程中起作用迟发,激活收缩蛋白和其他结构和代谢蛋白的基因表达(白金汉,2003; Bentzinger等人,2012)。
&NBSP; Myogenesis也受到许多转录因子和若干非编码RNA的调节,这些RNA在特定步骤中起作用,包括祖细胞(卫星)细胞对肌原性谱系和成肌细胞增殖,分化和融合的承诺(Yin et al,2013; Simionescu-Bankston和Kumar,2016)。使用培养的成肌细胞进行研究各种调节蛋白在肌发生中的作用的初步实验。在分化培养基中孵育时,存在分化成肌管的几种成肌细胞系(例如或,C2C12,L6,BC3H1和MM14)。这些细胞系也被用于建立肌管培养物以研究各种分子对肌管生长和萎缩的影响。然而,由于细胞的来源,培养条件和通过次数,结果往往存在一定程度的变异性。强烈推荐使用原代成肌细胞,因为它们没有永生化过程的特征和与活体的生理相关性。初级成肌细胞可以从新生儿或成年小鼠的骨骼肌中分离出来。然而,从新生儿肌肉分离成肌细胞的过程更复杂,因为它也需要percoll密度梯度离心(Dogra等人,2006)。一些研究者还使用荧光激活细胞分选(FACS)方法将原代成肌细胞从消化的肌肉组织中分离,特别是研究这些细胞的静止和活化的调节。然而,FACS分选是一种昂贵的方法,其需要几种阴性和阳性选择抗体和细胞分选机。此外,成肌细胞的产量通常较低,并且通过FACS技术在纯化的成肌细胞分离期间总是存在污染的机会。在我们的实验室,我们已经修改和标准化了以前发布的协议(Rando和Blau,1994),用于从成年小鼠的骨骼肌分离成肌细胞。该方案对于从成年小鼠的骨骼肌(Ogura等人,2015; Hindi和Kumar,2016)产生大量纯化的成肌细胞是非常有效的。可以通过免疫染色在未分化成肌细胞中表达的Pax7和MyoD蛋白来测定成肌细胞的纯度。此外,使用该方案分离的原代成肌细胞在分化培养基中孵育时有效地分化成多核肌管,并且可以通过相差显微镜或在分化肌细胞中表达的肌球蛋白重链(MyHC)免疫染色后容易地显现肌管(印度 et al。,2014; Bohnert等人,2016)。最后,像肌源性细胞系一样,纯化的原代成肌细胞可以在液氮或-80°C储存无限时间,并可以在需要时再生长。

关键字:成肌细胞, 骨骼肌, 肌细胞生成, MyoD, Pax7, 肌细胞生成素, 成肌分化, 后肢肌肉

材料和试剂

  1. 灭菌袋(Fisher Scientific,目录号:01-812-51)
  2. 100 x 20毫米 - 培养皿(康宁,目录号:430167)
  3. 6孔板(Corning,Falcon ®,目录号:353046)
  4. 1.5ml Eppendorf管(USA Scientific,目录号:1615-5510)
  5. 0.22μm过滤器(EMD Millipore,目录号:SLGP033RS)
  6. 15 ml无菌管(VWR,目录号:89004-368)
  7. 1毫升移液器尖端
  8. 石蜡膜
  9. 10 ml血清移液管(Santa Cruz Buitechnology,目录号:sc-200281)
  10. 70微米过滤器(Fisher Scientific,目录号:22-363-548)
  11. 50ml无菌管(VWR,目录号:89004-364)
  12. 30μm过滤器(Milteny Biotech,目录号:130-041-407)
  13. 0.45μm过滤器(EMD Millipore,目录号:SLHV033RS)
  14. 24孔板(Corning,Falcon ®,目录号:353047)
  15. 滑头注射器(BD,目录号:302833)
  16. 无菌细胞刮刀(Corning,Falcon ®,目录号:353085)
  17. 成年老鼠(Mus musculus; 6-8周龄)(见注释7和8)
  18. 2,2,2-三溴乙醇(Avertin)(Sigma-Aldrich,目录号:T48402)
  19. 0.25%胰蛋白酶 - 乙二胺四乙酸(EDTA)(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  20. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  21. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  22. 一抗抗体Pax7(小鼠)(Developmental Studies Hybridoma Bank,catalog number:Pax7)
  23. 一抗抗体MyoD(兔)(Santa Cruz Biotechnology,目录号:sc-304)
  24. 一抗抗体MyHC(小鼠)(Developmental Studies Hybridoma Bank,目录号:MF-20)
  25. 第二抗体山羊抗兔Alexa Fluor 488缀合物(Thermo Fisher Scientific,Invitrogen,目录号:A-11034)
  26. 第二抗体山羊抗小鼠Alexa Fluor 568缀合物(Thermo Fisher Scientific,Invitrogen,目录号:A-11004)
  27. 100%乙醇(Decon Labs,目录号:2701)
  28. Matrigel(康宁,目录号:354234)
  29. Dulbecco改良的Eagle's培养基(DMEM)高葡萄糖,丙酮酸(Thermo Fisher Scientific,Gibco TM,目录号:11995065)
  30. 胶原酶II(Worthington Biochemical,目录号:LS004176)
  31. 超纯水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  32. 青霉素 - 链霉素(Pen/Strep)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  33. N-2-羟乙基哌嗪-N-2-乙磺酸(HEPES)(1M)(Thermo Fisher Scientific,Gibco< sup>,目录号:15630080)
  34. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437028)
  35. 重组人成纤维细胞生长因子基因(bFGF)(PeproTech,目录号:100-18B)
  36. Tris碱(Fisher Scientific,目录号:BP152-5)
  37. F-10营养混合物(Thermo Fisher Scientific,Gibco TM,目录号:11550043)
  38. Dulbecco改良Eagle's培养基(DMEM)(ATCC,目录号:30-2002)
  39. 马血清(Thermo Fisher Scientific,Gibco TM ,目录号:26050088)
  40. 二甲基亚砜(DMSO)(Fisher Scientific,目录号:BP231-100)
  41. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  42. 100%Triton X-100(Fisher Scientific,目录号:BP151-500)
  43. 4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)(Sigma-Aldrich,目录号:D8417)
  44. 70%乙醇(见食谱)
  45. 10%Matrigel(见食谱)
  46. 胶原酶II(参见食谱)
  47. 消化培养基(参见食谱)
  48. 收集/洗涤/切碎溶液(参见食谱)
  49. 中和/隔离介质(见配方)
  50. 碱性成纤维细胞生长因子(bFGF)(参见食谱)
  51. 成肌细胞生长培养基(MGM)(参见食谱)
  52. 后隔离洗涤介质(参见食谱)
  53. 分化培养基(DM)(参见食谱)
  54. 冷冻介质(参见食谱)
  55. 4%多聚甲醛(PFA)(见配方)
  56. 0.3%Triton X-100(参见食谱)
  57. 10%Triton X-100(见配方)
  58. 阻塞解决方案(见配方)
  59. DAPI(见配方)

设备

  1. 高压釜
  2. 解剖工具:灭菌/高压灭菌剪刀和镊子
  3. 生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300 Series II,A2型)
  4. 用于1.5ml Eppendorf管的台式离心机(Eppendorf,型号:5424/5424 R)
  5. 用于15ml和50ml管的台式离心机(Eppendorf,型号:5702/5702 R/5702 RH)
  6. 加热培养箱振荡器(Eppendorf,New Brunswick TM ,型号:Excella E24)
  7. CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3578)
  8. 显微镜(Nikon Instruments,型号:Eclipse TE2000)
  9. 水浴(Thermo Fisher Scientific,型号:Isotemp TM数字控制水浴:215型,目录号:15-462-15Q)
  10. 500毫升瓶子

程序

分离和纯化小鼠后肢肌肉的原代成肌细胞的基本步骤如图1所示

图1.从小鼠后肢肌肉分离原代成肌细胞的一般程序的示意图。 MGM,成肌细胞生长培养基; FGF,成纤维细胞生长因子; DM,分化培养基。

  1. 在分离初级成肌细胞之前
    1. 在每个包含一把小剪刀和镊子的灭菌袋中高压灭菌两套工具(见注1和2)。
    2. 按照配方1,准备70%乙醇
    3. 按照配方2,准备10%Matrigel(见注3)。
    4. 按照方法3,制备胶原酶II(见附注4)
    5. 按照配方4,准备消化培养基(见注5和6)
    6. 按照配方5,准备收集/洗涤/切碎溶液。
    7. 按照配方6,准备中和/隔离介质(见注5)
    8. 按照方案8,准备成肌细胞生长培养基(MGM)(见注5)
    9. 通过倾倒并铺展10%Matrigel溶液以覆盖板区域,将100×20mm培养皿(每只小鼠一个板)涂覆。避免气泡。 30秒后,除去过量的基质胶溶液(如果保持无菌,可以收集多余的10%Matrigel并重新使用多次重复使用)。在生物安全柜(约20分钟)内,将无盖帽放在无菌空气流中干燥。一旦干燥,将盖子放在板上,并在室温下离开无菌生物安全柜,直到准备使用。没有必要打开生物安全柜的紫外灯。该板将用于在用胶原酶消化肌肉后分离和培养成肌细胞。
    10. 对于消化前的肌肉收集和洗涤,制备含有3ml /孔的收集/洗涤/切碎溶液的6孔板(只需要每只小鼠需要6孔板的4个孔)。
    11. 对于分离后的肌肉切碎,每只小鼠准备两个1.5ml Eppendorf管,每个包含500μl收集/洗涤/切碎溶液。

  2. 肌肉清除和清洗
    1. 在您的实验室中批准IACUC指南后,安乐死小鼠。为了该方案的目的,给予小鼠以250mg/kg的剂量腹膜内注射2,2,2-三溴乙醇,接着颈椎脱位。
    2. 在生物安全柜外部进行从小鼠分离后肢肌肉。对于该步骤,除了高压灭菌的解剖工具和乙醇喷雾之外,不需要无菌技术。用70%乙醇喷洒后肢,将鼠标朝上。
    3. 使用来自后肢的高压灭菌剪刀去除皮肤以暴露肌肉。
    4. 使用第一对高压灭菌的剪刀和镊子,从一个后肢收集胫骨前,腓肠肌,比目鱼,四头肌和伸肌腱长肌,并将它们放入含有收集/洗涤/切碎溶液的6孔板的一个孔中(视频1 )。

    5. 在另一个后肢重复上一步,将肌肉放在含有收集/清洗/切碎溶液的6孔板的第二口中(见注9)。
    6. 使用第二对高压灭菌镊子将肌肉转移到装有收集/洗涤/切碎溶液的6孔板的另外两个孔中。这是需要洗涤和去除孤立的肌肉上的任何粘着的头发或不想要的组织。
    7. 将盖子放在含有肌肉和收集/洗涤/切碎溶液的6孔板上,并用70%乙醇喷雾,然后将板转移到无菌生物安全柜。从此步骤开始,所有程序必须在生物安全柜内执行,以避免肌纤维的潜在污染。 

  3. 肌肉切碎/消化
    1. 使用第二对高压灭菌的镊子将洗涤的肌肉转移到两个1.5ml Eppendorf管(每个后肢肌肉组一个管),其含有收集/洗涤/切碎溶液,并使用第二对高压灭菌剪刀非常精细地切碎肌肉(参见附注10)。 ;
    2. 一旦肌肉被切碎,使用RTL离心机21,130 x g旋转30秒,将收集/洗涤/切碎溶液中的切碎的肌肉碎片分离。
    3. 将无菌生物安全柜内的纺管放在一边。
    4. 通过将1ml胶原酶II加入到先前制备的消化介质的基底中完成消化介质的制备(参见附注6)。使用0.22μm注射器过滤器将整个消化混合物过滤到15 ml管中
    5. 从含有肌肉的Eppendorf管中取出上清(步骤C2)。
    6. 将切碎的肌肉转移到消化培养基管中,切除无菌1 ml移液管尖端的尖端,以形成大孔以适应切碎的肌肉尺寸。
    7. 将少量消化培养基转移到Eppendorf管中以重新切碎肌肉,然后将混合物(切碎的肌肉+消化培养基)转移到原始的15ml消化培养基管(步骤C4)中。
    8. 重复步骤C7,直到所有切碎的肌肉从Eppendorf管转移到含有消化混合物的15ml管中。
    9. 关闭含有切碎肌肉和消化介质的15 ml管的盖子,并用Parafilm包裹盖子,以避免任何潜在的渗漏。
    10. 漩涡管5秒。
    11. 将管子水平放置在37℃的振荡器中,并以100rpm的速度摇动1小时,再用另外5秒的涡旋中途穿过(见注11)。
    12. 1小时后,混合应出现多云,肌肉块大部分消化很小(见附注12)
    13. 一旦消化完成,涡流管含有消化的肌肉混合物5秒
    14. 在室温下以1,400×g旋转管5分钟。
    15. 在无菌生物安全柜内,取出上清(消化介质)并弃去
    16. 使用10ml血清移液管将消化的肌肉沉淀重悬于7ml中和/分离培养基中。使用无菌10ml移液管将消化的肌肉组织颗粒上下移动20-30次。这确保大多数成肌细胞从肌肉组织释放。
    17. 将70μm过滤器放在50ml管上
    18. 用2 ml中和/隔离介质预先将70μm过滤器预湿。
    19. 使用10ml血清移液器收集重悬肌肉丸混合物(步骤C16),并通过预湿70μm过滤器。
    20. 用2ml中和/分离介质洗涤过滤器,以确保细胞粘附在过滤器上(在此步骤中大多数组织碎片将被除去)。
    21. 在15 ml管中,用1 ml中和/分离培养基预湿30μm过滤器
    22. 通过预湿30微米过滤器通过细胞混合物(先前通过70微米过滤器应变)。这一步将从成肌细胞中去除大的浸润细胞,如巨噬细胞
    23. 用1ml中和/分离介质清洗过滤器,以确保细胞不会粘附在过滤器上
    24. 含有细胞混合物的旋转管以1,400×g×5分钟。
    25. 去除上清液(中和/分离培养基),并将细胞沉淀重悬于10ml MGM + bFGF中
    26. 将细胞混合物种在10%Matrigel预涂的培养皿上。
    27. 在显微镜下观察细胞。在这一步,肌肉颗粒和细胞将会有一个不均匀的混合物(图2)。


      图2.在不同纯化阶段初次成肌细胞培养物的代表性相差显微镜图像在(A)0h,(B)的10%Matrigel包被的培养皿中接种后过滤消化的肌肉混合物, 24小时,(C)72小时。 (D)第一次预镀和(E)第二次预镀步骤后成肌细胞的外观。 F.在2-4次预镀步骤后,纯化成肌细胞培养物。刻度棒=20μm。蓝色箭头指向组织/细胞碎片;红色箭头指向成纤维细胞(大而扁平的三角形);绿色箭头指向成肌细胞(圆形或双轴纺锤形)。

    28. 将培养皿保持在37℃CO 2培养箱中72小时。在这个潜伏期间不要改变培养基。
    29. 72小时后,除了残留的组织碎片外,您还应该可以看到粘附的,小的,液滴形的成肌细胞和大的三角形成纤维细胞。
    30. 此时,电池已准备好进行第一次预镀(见附注13)。

  4. 成肌细胞纯化(预镀)
    1. 从菜中取出培养基。
    2. 使用10ml血清移液管,用后隔离培养基(含有1%Pen/Strep的F-10培养基)轻轻洗涤粘附细胞两次。
    3. 通过加入2ml 0.25%胰蛋白酶-EDA并将细胞置于37℃在CO 2培养箱中2分钟来分离细胞。
    4. 在显微镜下可视化,以确保所有细胞分离。
    5. 一旦细胞分离,加入10ml MGM + bFGF到盘中(见附注14)
    6. 将细胞混合物转移到非Matrigel包被的组织培养皿中,并在37℃在CO 2培养箱中放置45分钟。这将允许更大的细胞如成纤维细胞粘附在盘的底部,而大多数成肌细胞仍将悬浮在培养基中。
    7. 在这段时间里,用上面描述的方法涂上10%Matrigel的新培养皿
    8. 45分钟后,将所有上清液从预镀盘转移到新的Matrigel包被的培养皿中,并将盘放在CO 2培养箱中(见附注15)。
    9. 预镀步骤应每36-48小时进行一次,直至>达到98%的成肌细胞纯度(见附注16)。图2C-2E给出了成肌细胞分离的各个步骤中的代表性的对比图像。纯化的成肌细胞培养物图像如图2F所示。

  5. 培养成肌细胞
    1. 纯化的成肌细胞通过在成肌细胞生长培养基中孵育并每隔一天更换培养基而保持在培养物中。
    2. 不允许单元格达到> 70%融合(除非实验需要),因为这将导致成肌细胞的成熟前分化和融合。
    3. 根据需要将细胞分裂成多个10%Matrigel涂层的培养皿
    4. 成肌细胞可以收集在冷冻介质中并像任何其他培养细胞一样储存在液氮罐或-80°C冰箱中。初级成肌细胞一旦解冻,维持其增殖和分化能力,再次培养
  6. 将初级成肌细胞分化为肌管
    1. 具有类似于上述那些的10%Matrigel的涂层组织培养板
    2. 预涂基质胶板中的原始成肌细胞。为了获得优质的肌管,成肌细胞在加入分化培养基(DM)之前应该达到85-95%的汇合度。对于6孔组织培养板,我们需要每孔0.75-0.9×10 6个细胞;然而,该数量将根据细胞通道数而变化。低通道细胞的大小通常较小,因此使用范围的较高端实现所需的融合,而较高的传代细胞的细胞尺寸更大并且尺寸较大,所以需要细胞数量的较低端(参见附注17)。 />
    3. 第二天,用DM替代成肌细胞生长培养基(见附注18)。
    4. DM应每48小时更换一次。
    5. 成肌细胞在DM培养24-36小时内排列并融合。在48小时,可以清楚地看到小肌管,并且在72-96小时之间变得大而成熟
  7. 初级成肌细胞和肌管的免疫染色
    1. 从含有原始成肌细胞或肌管的6孔组织培养板中取出培养基,这些培养板粘附在孔的表面。
    2. 通过添加适量的4%PFA来覆盖细胞并孵育15分钟来固定粘附的细胞
    3. 去除4%的PFA,并用1x PBS轻轻洗涤细胞三次,每次5分钟
    4. 通过加入0.3%Triton X-100对细胞进行渗透,并在室温下孵育7分钟
    5. 去除0.3%Triton X-100,并用PBS轻轻洗涤细胞3次,每次5分钟
    6. 通过加入2%BSA溶液20分钟除去PBS并阻断细胞
    7. 在封闭溶液中制备一抗抗体混合物。
      注意:我们使用1:10稀释的Pax7抗体,1:200稀释的MyoD和1:200稀释的MF-20(即MyHC)抗体。
    8. 去除封闭溶液,并向每个孔中加入所需的一抗混合物
    9. 用Parafilm包裹板以防止一抗溶液的蒸发。
    10. 初级抗体孵育可以在室温下进行1-2小时,或在4℃下进行过夜。
    11. 一旦初次抗体孵育期完成,取出一抗溶液,用PBS洗涤细胞三次,每次5分钟
    12. 对于每个二级抗体,以1:1,500的浓度制备阻断溶液中的二抗混合物。
      注意:我们使用抗小鼠Alexa 568的红色荧光和抗兔Alexa 488进行绿色荧光。
    13. 用Parafilm包裹板以防止二次抗体的蒸发。
    14. 二次抗体孵育在室温下在黑暗中进行1小时
    15. 移除二抗溶液,用PBS洗涤细胞三次,每次5分钟
    16. 在PBS中准备DAPI工作解决方案。
    17. 从孔中取出PBS,并加入用于染色核的DAPI溶液
    18. DAPI孵育在黑暗中在室温下进行3分钟
    19. 取出DAPI,用PBS洗涤细胞三次,每次5分钟。
    20. 将PBS加入孔中。
    21. 细胞现在可以在荧光显微镜下显现。用抗Pax7,抗MyoD和DAPI染色后的成肌细胞培养物的代表性图示于图3中。用抗MyHC和DAPI染色后的分化肌管培养物图像示于图4中。


      图3.用于Pax7和MyoD的原代成肌细胞培养物的染色将在MGM中生长的原代成肌细胞培养物固定并对Pax7和MyoD蛋白进行免疫染色。核用DAPI进行复染。在这里介绍代表性的个人抗Pax7,抗MyoD和DAPI染色和合并的图像(三重染色)。比例尺= 20μm

      图4.肌球蛋白重链(MyHC)的主要肌管培养物的染色。将初级成肌细胞在DM中孵育24小时或48小时。将培养物固定并用MyHC蛋白免疫染色。核用DAPI进行复染。在这里介绍代表性的个人反MyHC和DAPI染色和合并的图像。比例尺= 20μm

数据分析

对于个别实验,样品量应通过功率分析来确定。我们大多数研究集中在研究调节因子对成肌细胞增殖和分化的影响,我们在4-5次重复中进行实验。为了研究肌原性分化,我们计算肌成纤维指数,这是MyHC染色的肌管在板中总核中的核的百分比。我们将数据呈现为平均值±标准偏差(SD)。我们使用配对或不配对的Student's -test来确定不同组之间的统计差异,类似于(0gura等人,2015; Hindi和Kumar,2016)。 A 0.05被认为具有统计学意义。

笔记

  1. 所有步骤除了后肢肌肉的初始分离应在无菌条件下进行。即使从安乐死的小鼠中分离后肢肌肉,强烈推荐使用高压消毒的解剖工具和喷雾乙醇。
  2. 一套将用于从小鼠收集肌肉,而另一套将在无菌生物安全柜下使用,以转移和切断肌肉。
  3. 不要让100%Matrigel达到RT,因为这会使其凝固,并形成一种浓缩的凝胶,如难以在DMEM中稀释的材料。
  4. 在所有批次中,沃辛顿胶原酶的粉末原料不具有相同的活性(即,单位为mg干重)。因此,每次准备胶原酶原料时,请仔细阅读原料粉末小瓶中存在的活性
  5. 所有介质应在37°C使用前加温。
  6. 当肌肉切碎并准备消化时,胶原酶只能加入,因为提前加入胶原酶会削弱其消化活性。
  7. 要从成人卫星细胞获得原代成肌细胞,小鼠必须≥6周龄。
  8. 为了获得最佳的初级成肌细胞,小鼠不应该≥8周龄。如果实验设置需要,则可以对年轻或老年小鼠进行该方案,然而可观察到肌原性产量和纯度的一些改变;年轻的小鼠会产生更多的成肌细胞,因为它们的肌肉含有促进肌肉发育和生长的活跃的增殖细胞。另一方面,较老的或患病的小鼠可能产生较少的成肌细胞,具有较低的纯度和较高的成纤维细胞含量。当从基因小鼠模型或经受各种实验程序的小鼠分离原代成肌细胞时,应考虑到类似的考虑。
  9. 在两分钟内肌肉收集应在5分钟内进行,以保持活力。
  10. 确保肌肉在这一步中精细切碎,因为这会影响成肌细胞的产量。
  11. 在这一点上观察消化肌肉混合物的外观很重要。肌肉片应看起来较小,消化介质应开始出现多云。如果肌肉片非常小,那么将下半年的消化时间减少到20分钟。
  12. 如果肌肉块的尺寸仍然较大,似乎没有完全消化,则将消化时间延长10分钟或直到消化完成。
  13. 如果细胞密度在分离后72小时非常低,此时不要预片,只需用F-10 + 1%PS洗涤菜,再用新鲜的MGM + bFGF再喂。
  14. bFGF以10ng/ml的浓度直接加入到培养皿中,或者可以以相似的浓度提前制备MGM + bFGF的混合物,但是如果适当地储存在4周时,这仅适用于1周°C。
  15. 有时,成肌细胞也会沉淀到预镀盘的底部,因此,如果大量的成肌细胞观察到重复预镀步骤较短的持续时间(约20分钟),那么第二天再加入培养基, 。
  16. 通常可以通过进行1-4次电镀重复来实现高成肌细胞纯度,但在某些情况下,这是不够的。如果成肌细胞似乎没有越来越多地主导着每个预镀步骤的文化,那么可以解决一种替代的纯化策略:
    1. 将您的文化分成多种培养皿,以便每道菜含有约10%融合的细胞混合物。
    2. 每个单元格应与其相邻单元格空间分开显示。
    3. 允许细胞持续生长,每隔一天更换米高梅。
    4. 每天应该观察源自单细胞的成肌细胞集落的形成。您还将观察到源自单个细胞的成纤维细胞菌落的形成
    5. 在倒置显微镜下,定位观察成纤维细胞集落的区域,并使用标记来标记该盘的底部。
    6. 在无菌生物安全柜下,使用无菌细胞刮刀物理擦拭成纤维细胞集落(注16e中标示)。
    7. 吸出培养基并用分离后的洗涤介质洗几次。
    8. 在笔记16f和16g中刮去和洗去成纤维细胞菌落后剩下的细胞(应该是主要成肌细胞集落)留下胰蛋白酶化。
    9. 将您的细胞置于新涂10%Matrigel菜中。
    10. 应该观察到高成肌细胞纯度。
  17. 避免在电镀分化过程中过度拥挤细胞,因为这将限制成肌细胞在融合之前伸长所需的空间,并对细胞施加额外的压力,可能导致细胞死亡和缺陷的肌管形成。
  18. 如果细胞在第二天不处于最佳汇合处,允许细胞增殖更长以达到期望的汇合度,然后再切换到DM。

食谱

  1. 70%乙醇
    将700ml 100%乙醇与300ml去离子水结合使用
  2. 10%Matrigel
    1. 解冻Matrigel在冰过夜
    2. 将10ml冷Matrigel溶于90 ml冷DMEM中
    3. 将15 ml 10%Matrigel每15 ml无菌管分批成多个管,以供以后使用
    4. 将10%Matrigel等分试样储存在-20°C长时间储存
    5. 为了经常使用,将10%Matrigel管储存在4°C
    注意:一旦从-20°C中取出并解冻,不要将10%Matrigel溶液重新冻结。
  3. 胶原酶II
    1. 通过将胶原酶II溶解在浓度为4000 U/ml的超纯水中来制备原料
    2. 将这种胶原酶II储备溶液分配到无菌Eppendorf管中的多个1ml等分试样中,以备以后使用
    3. 在-20°C储存等分试样
    4. 解冻胶原酶II即将使用前
    注意:避免重复的冻融循环,因为这将降低胶原酶II的酶活性。
  4. 消化介质
    10 ml消化培养基足以消化一只小鼠的后肢肌肉,如果使用较老的小鼠,可以修改体积。
    10 ml:
    1. 通过用100μlPen/Strep(1%)和250μlHEPES(2.5%)补充8.65ml DMEM(Gibco)来制备消化培养基。保持在37°C在水浴中直到准备使用
    2. 一旦肌肉被分离,切碎并准备消化,加入胶原酶II使终浓度达到400 U/ml。手动握手
    3. 过滤消解介质使用0.22μm过滤器
  5. 收集/洗涤/切碎溶液
    对于15ml,将150μlPen/Strep与14.85ml 1x PBS组合以获得1%Pen/Strep在PBS中的溶液
  6. 中和/隔离介质
    准备一个500毫升的中和介质:
    1. 从500毫升瓶中取出50ml的DMEM(Gibco),并在4℃下储存以备以后使用
    2. 将50ml过滤的FBS加入到现在含有450ml DMEM的DMEM瓶中,以达到10%FBS的浓度
    3. 将5 ml Pen/Strep加入到上述混合物中,以达到1%Pen/Strep浓度
    4. 将该库存存放在4°C
    5. 使用前温度要求在37°C以上
  7. 碱性成纤维细胞生长因子(bFGF)
    1. 用0.1%BSA稀释50μgbFGF(50μg/ml)于1ml过滤灭菌的5mM Tris(pH 7.6)中的溶液。
    2. 等分成小(50-100μl)并储存在-20°C或-80°C
    3. 解冻等份立即使用前
    注意:避免重复冻融循环。
  8. 成肌细胞生长培养基(MGM)
    准备一个500毫升的米高梅股票:
    1. 从500毫升的瓶子中取出100毫升的F-10培养基,并将去除的培养基储存在4℃,以进行任何潜在的使用
    2. 将100ml过滤的FBS加入到400ml F-10培养基中。加入5ml Pen/Strep溶液
    3. 将介质储存在4°C
    4. 使用前,请在37°C温热所需的量
    5. 在使用细胞培养基之前加入10ng/ml碱性成纤维细胞生长因子(bFGF)(注12)
    6. 每道菜需要10毫升米高梅
  9. 后隔离洗涤介质
    50 ml:
    补充49.5毫升具有500微升Pen/Strep的F-10培养基,以达到1%Pen/Strep浓度
    这可以在4°C储存。使用前温度37°C
  10. 分化培养基(DM)
    对于500毫升的库存:
    1. 从500毫升的瓶子中取出15毫升的DMEM(ATCC)
    2. 将10 ml过滤的马血清加入到DMEM瓶中
    3. 向上述混合物中加入5ml Pen/Strep,以达到1%Pen/Strep
      的浓度
    4. 将该库存存放在4°C
    5. 使用前温度要求在37°C以上
  11. 冷冻介质
    10 ml:
    将5 ml MGM(参见食谱8)与4 ml过滤的FBS(40%FBS)和1 ml DMSO(10%)结合使用
    这可以在4°C下储存,并在使用前加热
  12. 4%多聚甲醛(PFA)
    10 ml:
    1. 将0.4g PFA悬浮于10ml PBS中
    2. 以250rpm在60℃下摇动1-2小时直至完全溶解
    3. 允许在室温下冷却,并通过0.45μm注射器过滤器过滤
    4. 储存于4°C
    5. 为了获得最佳效果,如果存储超过2天
      ,请勿使用4%的PFA
  13. 0.3%Triton X-100
    1. 通过将1ml 100%Triton X-100与9ml PBS组合来制备10%Triton X-100的储备。
    2. 为了制备10ml 0.3%Triton X-100,加入300μl10%Triton X-100至9.7ml PBS
  14. 阻塞解决方案
    对于50ml:
    1. 将1g BSA溶于50ml PBS中
    2. 使用0.45μm注射器过滤器过滤器
    3. 这可以保存在-20°C以备将来使用
  15. DAPI
    10 ml:
    加入2μlDAPI储备溶液至10ml PBS,并使用0.45μm注射器过滤器过滤 DAPI的工作溶液可以在黑暗中保存在4°C

致谢

这项工作得到NIH拨款AR059810,AR068313和AG029623(对A. Kumar)和AR069985(到S. M.印地语)的资助。该协议已经从以前发表的文章中被修改和略微修改(Rando和Blau,1994; Hindi 等人,2014; Ogura等人,2015;印地语和Kumar ,2016)。

参考

  1. Bentzinger,CF,Wang,YX和Rudnicki,MA(2012)。  建立肌肉:分化调控肌发生。冷泉哈勃视野生物 4(2)。
  2. Bohnert,KR,Gallot,YS,Sato,S.,Xiong,G.,Hindi,SM and Kumar,A.(2016)。  抑制ER应激和解折叠蛋白反应途径导致癌症恶病质期间的骨骼肌消瘦。 FASEB J 30 9):3053-3068。
  3. Buckingham,M.,Bajard,L.,Chang,T.,Daubas,P.,Hadchouel,J.,Meilhac,S.,Montarras,D.,Rocancourt,D。和Relaix,F。(2003) 骨骼肌的形成:从体节到肢体。 J anat 202(1):59-68。
  4. Dogra,C.,Changotra,H.,Mohan,S。和Kumar,A。(2006)。凋亡的坏死因子样弱诱导物通过核因子-κB的持续激活和MyoD蛋白的降解来抑制骨骼肌发生。 > 281(15):10327-10336。
  5. 印度,SM和Kumar,A.(2016)。 TRAF6调节卫生干细胞自我更新和再生肌发生过程中的功能。 J Clin Invest 126(1):151-168。
  6. 印度,SM,Mishra,V.,Bhatnagar,S.,Tajrishi,MM,Ogura,Y.,Yan,Z.,Burkly,LC,Zheng,TS和Kumar,A。(2014)。 TWEAK-Fn14系统和PGC-1alpha在骨骼肌萎缩程序中的调节电路。/a> FASEB J 28(3):1398-1411。
  7. Ogura,Y.,Hindi,SM,Sato,S.,Xiong,G.,Akira,S。和Kumar,A.(2015)。 TAK1调节卫星干细胞体内平衡和骨骼肌修复。 6 6:10123。 />
  8. Rando,TA和Blau,HM(1994)。主要小鼠成肌细胞纯化,表征和移植用于细胞介导的基因治疗。 J Cell Biol <125>(6):1275-1287。
  9. Simionescu-Bankston,A.和Kumar,A.(2016)。卫星细胞和肌肉干细胞的生态位。生理学版93(1):23-67。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hindi, L., McMillan, J. D., Afroze, D., Hindi, S. M. and Kumar, A. (2017). Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice. Bio-protocol 7(9): e2248. DOI: 10.21769/BioProtoc.2248.
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María Cueva
UTPL
Hi, I'm a student still learning about the cell culture processes, and first of all I would like to thank you for this contribution on protocols like this, it is really helpful for students that want to learn, as I do. Since this is a protocol for mouse myoblast culture I would like to know if it is applicable for human myoblast culture, or if the difference only lies in the media used. Again, thank you very much for your response.
11/12/2017 3:07:47 PM Reply
Ashok Kumar
Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, USA

Hi Maria, We have used this protocol only for preparation of primary myoblasts from mouse skeletal muscle. We have not tried human samples therefore we can not say whether same protocol will work for human biopsies. You may want to follow this protocol for human muscle as well. Good luck! Ashok Kumar

11/13/2017 7:26:42 AM


Paul Hanavan
RenBio
Everything worked great using your protocol, until differentiation of the myocytes to myotubes. It seems as though they are fine for the first 48hrs in differentiation, but then quickly die en masse by 96-hours. Within 24hrs of adding differentiation media the cells are clearly beginning to line up and start to fuse, so I am pretty sure the cells are actually myoblasts (I have not yet stained to determine their identity). But can you comment on the differentiation medium? Your protocol uses 2% HS (as do most), but I have seen up to 10%. I have also heat-inactivated the HS, but your protocol does not indicate whether this is necessary or advisable.
Also, a recent protocol in isolating primary myofibers indicates that sodium pyruvate is essential for their survival - does your media contain sodium pyruvate (ours does not). Thanks very much for your thoughts on this issue.
7/24/2017 7:23:07 AM Reply
Sajedah Hindi
University of Louisville

Thank you for your inquiry Paul and we apologize for the delayed response. Following this protocol, myoblast alignment and primary fusion is seen at around 24h after addition of DM (as you described) and we usually see mature myotubes by 48-72 h. The down side with myotubes prepared from primary myoblast is that they are not sustainable for more than 48-72h after complete myotube formation, i.e 96-120h after the initial addition of DM. This is probably due to the continuous contraction that occurs once myotubes have matured which leads to their detachment from the culturing surface. If what you are observing is cell death prior to myotube formation or during the process of differentiation you first want to exclude the possibility of contamination which could be coming from the differentiation media or any of the other culturing components. Make sure that the horse serum is filtered prior to its addition to the DMEM and make sure that your horse serum is not old (use within 6 months of opening even if aliquots are frozen at -20°). If you are still experiencing the same problem assuming that you are following this protocol and using the referenced reagents, and assuming that you are confident that what you are culturing are myoblasts, one thing that comes to mind is the confluency of the cells before addition of DM. Too high confluency can lead to such results. First of all if the cells are too crowded, this will impose a stressful environment which may lead to their death. Add to that the increase in cell size and elongation that occurs during the alignment process during differentiation, so if there is not enough available room for the cells to comfortably elongate they will start pushing against each other which will lead to their detachment and death. Perhaps consider plating your cells at a lower confluence at the time of DM addition.
Regarding the horse serum in the DM, we have previously used it at 10% which also works fine, however we observed a delay in the differentiation kinetics. Also under such conditions we did not have to change the DM as frequently as the case in the 2%, other than that it worked fine.
You are doing right by heat inactivating your horse serum as it is advisable for better differentiation results, we didn’t mention that in the protocol because the horse serum that we use and is referenced in the protocol is already pre-heat inactivated however we probably should have included a note on that matter.
As far as the sodium pyruvate content, both the F-10 and DMEM that are referenced in this protocol and are used in preparation of MGM and DM respectively contain sodium pyruvate.
We hope this is helpful however let us know if you have any other concerns or your current ones aren’t properly addressed.

8/12/2017 7:22:53 PM