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Jun 2018

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Isolation and Culture of Single Myofiber and Immunostaining of Satellite Cells from Adult C57BL/6J Mice
成年C57BL/6J大鼠单个肌纤维的培养分离和卫星细胞的免疫染色   

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Abstract

Myofiber isolation followed with ex vivo culture could recapitulate and visualize satellite cells (SCs) activation, proliferation, and differentiation. This approach could be taken to understand the physiology of satellite cells and the molecular mechanism of regulatory factors, in terms of the involvement of intrinsic factors over SCs quiescence, activation, proliferation and differentiation. Single myofiber culture has several advantages that the traditional approach such as FASC and cryosection could not compete with. For example, myofiber isolation and culture could be used to observe SCs activation, proliferation and differentiation at a continuous manner within their physiological “niche” environment while FACS or cryosection could only capture single time-point upon external stimulation to activate satellite cells by BaCl2, Cardiotoxin or ischemia. Furthermore, in vitro transfection with siRNA or overexpression vector could be performed under ex vivo culture to understand the detailed molecular function of a specific gene on SCs physiology. With these advantages, the physiological state of SCs could be analyzed at multiple designated time-points by immunofluorescence staining. In this protocol, we provide an efficient and practical protocol to isolate single myofiber from EDL muscle, followed with ex vivo culture and immunostaining.

Keywords: Myofiber isolation and culture (肌纤维的分离和培养), Muscle satellite cells (肌卫星细胞), Immunofluorescence staining (免疫荧光标记)

Background

Satellite cells are considered as an adult stem cell because they maintain self-renew and remarkable postnatal regenerative potential of skeletal muscle (Collins et al., 2005). SCs are located between the basal lamina and the plasma lemma of myofibers (MAURO, 1961). Here, to isolate the single myofiber, EDL muscle is digested with the collagenase to release the connective tissue and completely dissociate the connective tissue between fibers. In 1986, it was reported that the proliferation of satellite cells on single muscle fibers were isolated from adult rats and were cultured in cell culture plate (Bischoff, 1986). It was further developed into a tissue culture system that reliably permits isolation of intact, living, single muscle fibers with associated satellite cells from predominantly fast- and slow-twitch muscles of rat or mouse (Bekoff and Betz, 1977a and 1977b; Rosenblatt et al., 1995). There are two main approaches to study SCs: single myofiber culture and primary myoblast culture prepared from mononucleated cells dissociated from whole muscle (Danoviz and Yablonka-Reuveni, 2012). Even though, primary myoblast could be split and passaged multiple times, these primary myoblasts retain in proliferation or differentiation states. Freshly isolated single myofiber allows satellite cells to stay beneath the basal lamina at quiescent state, followed with activation by either internal environment or external factors. There are several improvements for myofiber isolation and culture in recent years (Danoviz and Yablonka-Reuveni, 2012; Pasut et al., 2013; Gallot et al., 2016; Lim et al., 2018). However, the difficulty of myofiber isolation and ex vivo culture prevent further application of this reliable and practical method. In the current protocol, we optimized reagents used for single myofiber isolation and improved procedures to make it even more simple and easy to repeat from hand to hand. Since satellite cells are sensitive to various factors, we describe a relatively simple, detailed and efficient approach to isolate and culture single myofiber. Based on our protocol, state and function of satellite cells could be analyzed from mice with different genotypes. Different manipulation such as transfection, and drug treatment on myofibers, followed with the downstream procedures including but not limited to myofiber transplantation and immunostaining could be performed. These additional manipulations could not be performed in these approaches such as FASC or cryosection.

Materials and Reagents

  1. Pipette tips (Jet Biofil, catalog number: PMT950200)
  2. 15 ml tube (TrueLine, catalog number: TR2011)
  3. 60 mm Petri Dish, Sterile (Jet Biofil, catalog number: 62060-60)
  4. 24-well Plates, Sterile (Corning, Costar, catalog number: CLS3524-100EA)
  5. Glass Pasteur Pipettes (Thermo Fisher, catalog number: 1367820A) (Figure 1e-g)
  6. Rubber bulbs (Thermo Fisher, catalog number: 1951F15) (Figure 1h)
  7. Diamond Pen (XGE, used to cut glass Pasteur pipettes) (Figure 1d)
  8. 1 ml Transfer Pipette, sterile (Jet Biofil, catalog number: 25001) 
  9. Syringe Filters (PTFE, 0.22 μm, 30 mm, Sterile) (Jet Biofil, catalog number: 29525)
  10. Microscope glass slide (CITOTEST, catalog number: 70179000)
  11. Microscope cover slide (24 x 50 mm) (CITOTEST, category number: 10212450C)
  12. Adult C57BL/6J mice at 8-10-week old
  13. Glycine (Sangon Biotech, catalog number: A610235, storage temperature: room temperature)
  14. Phosphate buffered saline (PBS, Sigma-Aldrich, catalog number: P5368-10PAK, storage temperature: room temperature)
  15. Triton X-100 (Sangon Biotech, catalog number: A600198, storage temperature: room temperature)
  16. Tween-20 (Sangon Biotech, catalog number: A100777, storage temperature: room temperature)
  17. Horse serum (short term storage: 4 °C; long term storage: -20 °C) (Thermo Fisher, catalog number: 16050122)
  18. Fetal bovine serum (short term storage: 4 °C; long term storage: -20 °C) (Trinity, catalog number: 010101)
  19. GibcoTM Sodium Pyruvate 100 mM Solution (Life Technologies, catalog number: 11360070, storage temperature: 4-8 °C)
  20. GibcoTM Penicillin-Streptomycin, liquid (Life Technologies, catalog number: 15140-122, storage temperature: 4-8 °C)
  21. Ethylene Diamine Tetraacetic Acid (EDTA, Solarbio, catalog number: E8040, storage temperature: 4-8 °C)
  22. GibcoTM Dulbecco's modified Eagle medium ( Life Technologies, catalog number: C11995500BT, storage temperature: 4-8 °C)
  23. Bovine Serum Albumin (IgG-Free, Protease-Free, Jackson ImmunoResearch, catalog number: 000-001-162, storage temperature: 4-8 °C)
  24. FluoroshieldTM with DAPI (Sigma-Aldrich, catalog number: F6057, storage temperature: 4-8 °C)
  25. Primary antibody anti-MyoD (Sigma-Aldrich, catalog number: M6190, storage temperature: 4-8 °C)
  26. Primary antibody anti-Pax7 (mouse) (Developmental Studies Hybridoma Bank, catalog number: pax7, storage temperature: 4-8 °C)
  27. Secondary antibodies:
    1. Goat anti-Rabbit IgG2a Alexa Fluor® 488 (Thermo Fisher Scientific, catalog number: A-21131, 4-8 °C)
    2. Goat anti-Mouse IgG Alexa Fluor® 546 (Thermo Fisher Scientific, catalog number: A-21123, 4-8 °C)
  28. Collagenase Type 1 (Worthington Biochemical, catalog number: LS004194, storage temperature: -20 to -30 °C)
  29. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148, storage temperature: 4-8 °C )
  30. Goat serum (Thermo Fisher, catalog number: 31872, storage temperature: -20 to -30 °C)
  31. EDTA (Solarbio, catalog number: E8040)
  32. Chicken embryo extract (C3999, Biomol)
  33. Washing media (see Recipe 1)
  34. Collagenase solution (see Recipe 2)
  35. 4% Paraformaldehyde (see Recipe 3)
  36. Culture media (see Recipe 4)
  37. Blocking buffer (see Recipe 5)

Equipment

  1. Pipettes (Thermo scientific, Finnpipette F3)
  2. Surgical instruments (including a pair of scissors, a pair of fine scissors and a tweezer) (Figure 1a, 1b and 1c)
  3. Cell culture hood (AIRTECH, model: BSC-1004IIA2)
  4. Mice dissecting table
  5. 4 °C fridge
  6. Centrifuge (Thermo Fisher, catalog number: 75002420)
  7. Water bath (DK-8AX)
  8. Stereoscopic microscope (Mshot, OLYMPUS SZ61)
  9. Heating pad (DeiuxeHeart Mat, model: MDH10)
  10. CO2 incubator (Thermo Fisher Scientific, model: 371)
  11. Laser scanning confocal microscope (Carl Zeiss, model: LSM 700)

Procedure

  1. Preparation
    1. Cut the Glass Pasteur Pipettes (Figure 1e) to a small curved (Figure 1f) and a large (Figure 1g) pipette with a diamond pen (Figure 1d). Polish the terminal with an ethanol flame to smooth the edge. Coat pipettes with undiluted horse serum.
    2. Coat two 6-cm cell culture dishes (2 ml for each dish) and a 24-well plate (400 μl for each well) with undiluted horse serum until the entire bottom of plates was covered, followed with incubation for 1 min under a cell culture hood, and then transfer the horse serum back to the original tube and store in a 4 °C fridge. 
    3. Uncover the plates and air-dry under a cell culture hood for 1 h.
    4. Cover the plates and keep them at room temperature. It is better to coat the plate on the same day of the experiment. 
    5. Set the temperature of a water bath to 37 °C.
    6. Prepare washing media (following Recipe 1 and then keep it in a 37 °C water bath). In general, 50 ml washing media is enough for up to 200 myofiber isolation from one EDL muscle.
    7. Prepare collagenase solution (following Recipe 2 and then keep it in a 37 °C water bath). Each EDL requires ~1 ml dilated collagenase solution (final concentration: 0.2% w/v) in a 15 ml tube.
    8. Thaw the 4% paraformaldehyde (following Recipe 3).
    9. Plug in the heating pad.


      Figure 1. Instruments used for EDL muscle dissection and myofiber isolation. a. Tweezer; b. Scissor; c. Fine scissor; d. Diamond pen; e. Glass Pasteur Pipette; f. Small curved pipette; g. Large pipette; h. Rubber bulbs.

  2. EDL Muscle Dissection (Video 1)

    Video 1. Procedures to isolate EDL muscle. This video was made at Guangdong Institute of Microbiology according to guidelines from the Guangdong Institute of Microbiology on Animal Care and approved by the Animal Research Ethics Board of Guangdong Institute of Microbiology under protocol [GT-IACUC201704071].

    1. Anesthetize the experimental mice with CO2.
    2. Cut the skin below the ankle.
    3. Cut through the skin along the inner leg.
    4. Expose the muscle fascia and patella by pulling the skin up (Figure 2A).
    5. Tore the tibialis anterior (TA) muscle fascia with a tweezer.
    6. Tear off vastus lateralis over the knee, expose and cut off the origin of TA and extensor digitorum longus (EDL) muscle (Figure 2B).
    7. Separate the tendon of TA muscle using a tweezer along the inner margin of the shank. Snip the tendons with the fine scissor (Figure 2C). Gently pull it up to separate the TA muscle from the bone and expose the entire EDL muscle (Figure 2D).
    8. Cut the tendon of EDL muscle by snipping two-thirds of the lower ankle, Use one tip of the forceps to separate EDL muscle gently (Figure 2E).
    9. Place the EDL muscle in a 15 ml tube with the prewarmed collagenase solution and place it in a 37 °C water bath to incubate for 60 to 80 min until myofibers are loose but not in crimped (it depends on the efficacy of collagenase and conditions of mice).
    10. Dissect the other EDL muscle following Steps B1-B9.


      Figure 2. Procedures to isolate the EDL muscle. Pictures presented here depict (A) TA muscle after the skin was removed, (B) Origin of TA and EDL muscle, (C) Distal tendon of TA muscle, (D) EDL muscle after TA muscle was removed, (E) EDL muscle. a. vastus lateralis; b. patella; c. tendon of EDL muscle; d. tendon of TA muscle; e. origin of TA muscle; f. origin of EDL muscle; g. tendon of TA muscle.

  3. Single Myofiber Isolation (Video 2)

    Video 2. Procedures to isolate myofiber from EDL muscle. This video was made at Guangdong Institute of Microbiology according to guidelines from the Guangdong Institute of Microbiology on Animal Care and approved by the Animal Research Ethics Board of Guangdong Institute of Microbiology under protocol GT-IACUC201704071.

    1. Transfer the digested EDL muscle to a horse serum coated 6-cm Petri dish with 5 ml prewarmed washing medium in it using the prepared big Glass Pasteur Pipette.
    2. Place the 6-cm Petri dish on the heating pad. Inhale an appropriate amount of solution from the dish. Hold the Petri dish with the right hand to adjust the position of dish. Flush the EDL muscle continuously to rotate it along its long pivots until myofibers are naturally released from EDL muscle.
    3. When a single muscle fiber is released, use a small bent glass Pasteur pipette to transfer it into a well of 24-well plate covered with 200 μl washing media. Repeat Steps C1 and C2 until the number of myofibers meets your requirement. It is recommended that the myofibers be collected within 1-h.
    4. Wash each well with 500 µl washing media three times before adding 500 µl culture media (following Recipe 4).
    5. Place the 24-well plate into a CO2 incubator at 37 °C.

  4. Fixation
    1. When myofibers are cultured for 0/24/48/72-h or other different designated length of time, remove as much culture media as possible (leave the minimum amount of culture media to avoid myofibers’ exposure to the air).
    2. Fix myofibers with 500 μl 4% PFA for 10 min at room temperature.
    3. Aspirate the fixation buffer, and add 500 μl 50 mM glycine to incubate for 10 min to quench the fixation at room temperature.
    4. Wash myofibers with PBS for 3 times at room temperature. The fixed myofibers can be stored in a 4 °C fridge for up to a week before immunostaining (Figure 3).


      Figure 3. Images of isolated single myofiber after cultured for 0/24/48/72 h. Scale bars = 100 μm.

  5. Immunostaining
    1. Permeabilize myofibers with Triton X-100 (0.5% v/v) diluted in PBS for 10-min, followed by a 5-min wash in PBS for 3 times.
    2. Prepare blocking buffer as Recipe 5. Centrifuge the blocking buffer at 16,000 x g for 10 min in case there was any residue precipitate from goat serum. Incubate fibers with 400 μl blocking solution for 1 h at room temperature.
    3. Incubate with 200 μl blocking buffer containing primary antibodies of Pax7 (1:100) or MyoD (1:300) overnight at 4 °C.
    4. After night incubation with primary antibody, wash myofibers 3 times for 5-min with PBS containing 0.5% Tween 20 to remove any unbound antibody.
    5. Incubate with 200 μl blocking buffer containing appropriate secondary antibody (1:500) for 1-h at room temperature protected from light.
    6. After 1-h incubation with secondary antibody, wash 3 times at 5 min per wash with PBS containing 0.5% Tween 20.
    7. Transfer each fiber to a glass slide and drain the remaining liquid.
    8. Mount the glass slide with DAPI-containing mounting medium. Cover with a cover slide and dry the glass slide for at least 10 min before imaging.
    9. Observe myofibers under a fluorescent microscope and capture images of immunostained myofibers (Figure 4).


      Figure 4. Representative images of single myofiber culture harvested at T0, 24, 48 and 72-h. Single myofiber with satellite cells or clusters were fixed and immunostained with antibodies against Pax7 or MyoD. Red for Pax7+ satellite cells. Purple for MyoD+ satellite cells. Blue for nucleus. Scale bar = 100 μm.

Data analysis

Based on our protocol, around ~200 myofibers could be isolated from each EDL muscle. The average number of Pax7+ satellite cells on each myofiber at T0 is 6-8 and the MyoD+ satellite cells could be barely detected at T0. After 24-h culture, the number of MyoD+ satellite cells is increased upon satellite cells activation. Proliferated satellite cells could be observed at 48-h timepoint. The satellite cells cluster could be observed after 72-h culture (Figure 4).

Notes

  1. Avoid bubbles when coating plates with horse serum.
  2. Do not let tweezers or scissors touch the EDL muscle.
  3. When using the bent plastic dropper to transfer muscle fiber, single myofiber should be inhaled along one end of the muscle fiber to avoid snapping the muscle.
  4. When transferring myofibers, insert the nozzle below the liquid level to prevent the muscle fibers from being exposed to air.
  5. Avoid bubbles when covering glass slips.

Recipes

  1. Washing media
    DMEM supplemented with:
    10% fetal bovine serum
    2% sodium pyruvate
    1% Penicillin-Streptomycin
    2 mM EDTA
    Filter the solution through a 0.2 μm filter (optional, to remove any debris from serum)
  2. Collagenase solution
    DMEM supplemented with 0.2% Collagenase Type 1
  3. 4% paraformaldehyde
    1. Dissolve 4 g paraformaldehyde powder in 100 ml PBS. 
    2. Heat the solution to 65 °C until paraformaldehyde completely dissolved
    3. Filter the solution through a 0.2 μm filter and store at -20 °C
  4. Culture media
    DMED supplemented with 20% fetal bovine serum
    1% chicken embryo extract
    1% Penicillin-Streptomycin
    Filter the solution through a 0.2 μm filter
  5. Blocking buffer
    PBS supplemented with:
    3% BSA
    0.5% Tween 20
    5% goat serum (added right before the blocking procedure)

Acknowledgments

This work was support by ‘GDAS’ Project of Science and Technology Development (Grant No. 2018GDASCX-0806) to Liwei Xie.
Author contribution: LW. X. designed the experiment and collected the grant for present study. LW. X., SJ. C., HR. D. and XP. Y. developed and optimized the methodology. SJ. C. collected and analyzed the data. SJ. C. drafted the manuscript and LW. X. proofread the manuscript.

Competing interests

The authors declare that there is no conflict of financial or research interest.

Ethics

C57BL/6J mice were raised at the SPF animal facility of Guangdong Institute of Microbiology (GDIM) in a 12/12 dark-light cycle with ad lithium free access to food and water. The animal protocol was proved by the Institute Animal Care Use Committees of GDIM (Permission #: GT-IACUC201704071).

References

  1. Bekoff, A. and Betz, W. (1977a). Properties of isolated adult rat muscle fibres maintained in tissue culture. J Physiol 271(2): 537-547.
  2. Bekoff, A. and Betz, W. J. (1977b). Physiological properties of dissociated muscle fibres obtained from innervated and denervated adult rat muscle. J Physiol 271(1): 25-40.
  3. Bischoff, R. (1986). Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115(1): 129-139.
  4. Collins, C. A., Olsen, I., Zammit, P. S., Heslop, L., Petrie, A., Partridge, T. A. and Morgan, J. E. (2005). Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122(2): 289-301.
  5. Danoviz, M. E. and Yablonka-Reuveni, Z. (2012). Skeletal muscle satellite cells: background and methods for isolation and analysis in a primary culture system. Methods Mol Biol 798: 21-52.
  6. 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.
  7. Lim, C. L., Ling, K. H. and Cheah, P. S. (2018). Isolation, cultivation and immunostaining of single myofibers: An improved approach to study the behavior of satellite cells. J Biol Methods 5(1): e87.
  8. Mauro, A. (1961). Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9: 493-495.
  9. 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.
  10. 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.

简介

肌纤维分离随后进行离体培养可以重现并可视化卫星细胞(SCs)的激活,增殖和分化。这种方法可以用来理解物理学单因素培养具有几个优势和优势,SCOC因素参与了激活因子。 SCs在其生理“生态位”环境中以连续方式激活,增殖和分化,而FACS或冷冻切片只能通过外部刺激捕获单个时间点以通过BaCl 2 激活卫星细胞。此外,用siRNA或过表达载体进行体外转染可在下进行利用这些优点,可以通过免疫荧光染色在多个指定的时间点分析SCs的生理状态从EDL肌肉中分离单个肌纤维,然后进行离体培养和免疫染色的有效且实用的方案。
【背景】卫星细胞被认为是一种成体干细胞,因为它们保持自我更新和骨骼肌的显着后势潜力(Collins et al。,2005).SCS位于基底层和血浆利马之间。在这里,为了分离单个肌纤维,EDL肌肉被胶原酶消化以释放结缔组织并完全解离结缔组织之间的结缔组织。1986年,据报道其他的增殖。它进一步发展成组织培养系统,其可以从成年大鼠中分离并在细胞培养板中培养(Bischoff,1986)。 Rosenblatt et al。,1995)。研究SC有两种主要方法:单一肌纤维培养和p。甚至肌肉,原代成肌细胞也可以从传代细胞中分离出来,从整个细胞中分离出来(Danoviz和Yablonka-Reuveni,2012)。在静息状态下,我的病情有几个方面,其次是内部环境或外部因素激活。多年来肌纤维分离和培养有几项改进(Danoviz和Yablonka-Reuveni,2012; Palesut。 ,2013; Gallot et al。,2016; Lim et al。,2018。)然而,肌纤维分离和离体的难度<自卫星以来,我们优化了用于单一肌纤维分离的试剂和改进的程序,使其更加简单,易于手工复制。根据我们的协议,可以从具有不同因子的小鼠中分析卫星细胞的状态和功能,如所描述的对各种因素敏感,我们描述了相对简单,详细和有效的分离和培养方法。这些添加处理不能进行,这些添加处理不能进行,这些额外的处理不能在下游程序中进行,包括但不限于下游程序。

关键字:肌纤维的分离和培养, 肌卫星细胞, 免疫荧光标记

材料和试剂

  1. 移液器吸头(Jet Biofil,目录号:PMT950200)
  2. 15毫升管(TrueLine,目录号:TR2011)
  3. 60毫米培养皿,无菌(Jet Biofil,目录号:62060-60)
  4. 24孔板,无菌(Corning,Costar,目录号:CLS 3524-100EA)
  5. 玻璃巴斯德吸管(Thermo Fisher,目录号:1367820A)(图1e-g)
  6. 橡胶灯泡(Thermo Fisher,目录号:1951F15)(图1h)
  7. 金刚笔(XGE,用于切割玻璃巴斯德吸管)(图1d)
  8. 1毫升转移移液管,无菌(Jet Biofil,目录号:25001)&nbsp;
  9. 注射式过滤器(PTFE,0.22μm,30 mm,无菌)(Jet Biofil,目录号:29525)
  10. 显微镜载玻片(CITOTEST,目录号:70179000)
  11. 显微镜盖玻片(24 x 50 mm)(CITOTEST,类别编号:10212450C)
  12. 8-10周龄的成年C57BL / 6J小鼠
  13. 甘氨酸(Sangon Biotech,目录号:A610235,储存温度:室温)
  14. 磷酸盐缓冲盐水(PBS,Sigma-Aldrich,目录号:P5368-10PAK,储存温度:室温)
  15. Triton X-100(Sangon Biotech,产品目录号:A600198,储存温度:室温)
  16. Tween-20(Sangon Biotech,目录号:A100777,储存温度:室温)
  17. 马血清(短期储存:4°C;长期储存:-20°C)(Thermo Fisher,目录号:16050122)
  18. 胎牛血清(短期储存:4°C;长期储存:-20°C)(Trinity,目录号:010101)
  19. GibcoTM丙酮酸钠100 mM溶液(Life Technologies,目录号:11360070,储存温度:4-8°C)
  20. Gibco TM 青霉素 - 链霉素,液体(Life Technologies,目录号:15140-122,储存温度:4-8°C)
  21. 乙二胺四乙酸(EDTA,Solarbio,目录号:E8040,储存温度:4-8°C)
  22. Gibco TM Dulbecco改良Eagle培养基(Life Technologies,目录号:C11995500BT,储存温度:4-8°C)
  23. 牛血清白蛋白(无IgG,无蛋白酶,Jackson ImmunoResearch,目录号:000-001-162,储存温度:4-8°C)
  24. 具有DAPI的Fluoroshield TM(Sigma-Aldrich,目录号:F6057,储存温度:4-8℃)
  25. 一抗抗MyoD(Sigma-Aldrich,目录号:M6190,储存温度:4-8°C)
  26. 一抗抗Pax7(小鼠)(Developmental Studies Hybridoma Bank,目录号:pax7,储存温度:4-8°C)
  27. 二抗:
    1. 山羊抗兔IgG2a Alexa Fluor ® 488(Thermo Fisher Scientific,目录号:A-21131,4-8°C)
    2. 山羊抗小鼠IgG Alexa Fluor ® 546(Thermo Fisher Scientific,目录号:A-21123,4-8°C)
  28. 胶原酶1型(Worthington Biochemical,目录号:LS004194,储存温度:-20至-30°C)
  29. 多聚甲醛(Sigma-Aldrich,目录号:P6148,储存温度:4-8°C)
  30. 山羊血清(Thermo Fisher,目录号:31872,储存温度:-20至-30°C)
  31. EDTA(Solarbio,目录号:E8040)
  32. 鸡胚提取物(C3999,Biomol)
  33. 洗涤介质(见食谱1)
  34. 胶原酶溶液(见配方2)
  35. 4%多聚甲醛(见配方3)
  36. 文化媒体(见食谱4)
  37. 阻塞缓冲区(见第5条)

设备

  1. 移液器(Thermo scientific,Finnpipette F3)
  2. 手术器械(包括一把剪刀和一把剪刀和一把镊子)(图1a,1b和1c)
  3. 细胞培养罩(AIRTECH,型号:BSC-1004IIA2)
  4. 小鼠解剖表
  5. 4°C冰箱
  6. 离心机(Thermo Fisher,目录号:75002420)
  7. 水浴(DK-8AX)
  8. 立体显微镜(Mshot,OLYMPUS SZ61)
  9. 加热垫(DeiuxeHeart Mat,型号:MDH10)
  10. CO 2 培养箱(Thermo Fisher Scientific,型号:371)
  11. 激光扫描共聚焦显微镜(Carl Zeiss,型号:LSM 700)

程序

  1. 准备
    1. 用未稀释的切割和粘贴移液器(图1e)涂抹移液管到小弯曲(图1f)和大(图1g)马血清。
    2. 用未稀释的马血清涂覆两个6-cm细胞培养皿(每个培养皿2ml)和24孔板(每孔400μl)直至覆盖整个底部,然后在细胞培养基下孵育1分钟。培养罩,然后将马血清转移回原管并储存在4°C的冰箱中。&nbsp;
    3. 揭开平板并在细胞培养罩下风干1小时。
    4. 最好在实验的同一天涂上盘子。&nbsp;
    5. 将水浴的温度设定为37°C。
    6. 准备洗涤介质(按照配方1,然后将其保存在37°C水浴中)。通常,50 ml洗涤介质足以从一个EDL肌肉中分离出200个肌纤维。
    7. 制备大肠杆菌胶原溶液(按照配方2,然后将其保持在37℃水浴中)。每个EDL在15ml管中需要~1ml稀释的胶原溶液(终浓度:0.2%w / v)。
    8. 解冻4%多聚甲醛(遵循配方3)。
    9. 插上加热垫。


      图1.用于EDL肌肉解剖和肌纤维分离的仪器。 a。镊子; b。剪刀; c。精细剪刀; d。钻石笔; e。玻璃巴斯德吸管; f。小弯曲移液管; g。大型移液器; h。橡胶灯泡。

  2. EDL肌肉解剖(视频1)


    视频1.分离EDL肌肉的程序。本视频由广东省微生物研究所根据广东省动物保健微生物研究所的指导制作,并经广东省微生物研究所动物研究伦理委员会批准。根据议定书[GT-IACUC 201704071]。

    1. 用CO 2 麻醉实验小鼠。
    2. 切开脚踝下方的皮肤。
    3. 沿着内腿切开皮肤。
    4. 拉起皮肤露出肌肉筋膜和髌骨(图2A)。
    5. 用镊子敲打胫骨前肌(TA)肌肉筋膜。
    6. 切除膝盖上的股外侧肌,暴露并切断TA和趾长伸肌(EDL)肌肉的起源(图2B)。
    7. 用细剪刀剪断肌腱(图2C)。轻轻拉起肌腱,将TA肌肉与骨骼分开,露出整个身体的EDL肌肉(图2D) )。
    8. 通过剪断下踝的三分之二切割EDL肌腱,使用镊子的一个尖端轻轻地分开EDL肌肉(图2E)。
    9. 将EDL肌肉置于带有预热胶原酶溶液的15 ml管中,置于37°C水浴中孵育60至80分钟,直至肌纤维松散但未卷曲小鼠)。
    10. 在步骤B1-B9之后解剖其他EDL肌肉。


      图2.分离EDL肌肉的程序。此处呈现的图片(A)去除皮肤后的TA肌肉,(B)TA和EDL肌肉的起源,(C)TA肌肉的远端肌腱B. Patella; c.EDL肌肉的肌腱; d.TA肌肉的肌腱; e.TA肌肉的起源; f。(D)去除TA肌肉后的EDL肌肉,(D)EDL肌肉。 EDL肌肉的起源; g.TA肌肉的肌腱。

  3. 单肌纤维隔离(视频2)


    视频2.将肌纤维与EDL肌肉隔离的程序。该视频是根据广东省动物保健微生物研究所的指导方针在广东省微生物研究所制作的,并经动物研究伦理委员会批准。根据方案GT-IACUC 201704071的微生物学。

    1. 将消化的EDL肌肉转移至马血清涂覆的6cm培养皿中,其中加入5ml预热的洗涤介质,使用制备的大玻璃巴斯德吸管。
    2. 用右手握住培养皿调整培养皿的位置。连续冲洗EDL肌肉,沿长轴旋转,直到加热垫上的6厘米培养皿。肌纤维自然地从EDL肌肉中释放出来。
    3. 重复步骤C1和C2,使肌纤维的数量符合您的要求。许多肌纤维在释放时符合肌纤维的状况,并使用一个小的弯曲玻璃巴斯德吸管将其转移到24孔的井中在1小时内收集肌纤维。
    4. 在加入500μl培养基之前,用500μl洗涤培养基洗涤每个孔三次(遵循配方4)。
    5. 将24孔板置于37℃的CO 2 培养箱中。

  4. 固定术
    1. 当肌纤维培养0/24/48 / 72-h或其他不同的指定时间长度时,尽可能多地去除培养基(留下最少量的培养基以避免肌纤维暴露在空气中)。
    2. 在室温下用500μl4%PFA固定肌纤维10分钟。
    3. 吸出固定缓冲液,加入500μl50mM甘氨酸孵育10分钟,在室温下淬灭固定。
    4. 在免疫染色前,固定的肌纤维可以储存在4°C的冰箱中长达一周(图3)。


      图3.培养0/24/48/72 h后分离的单个肌纤维的图像。比例尺=100μm。

  5. 免疫染色
    1. 用在PBS中稀释10分钟的Triton X-100(0.5%v / v)渗透肌纤维,然后在PBS中洗涤5分钟3次。
    2. 制备封闭缓冲液作为配方5.将封闭缓冲液以16,000 x g 离心10分钟,以防从山羊血清中接收任何残留物。在室温下用400μl封闭溶液孵育纤维1小时。
    3. 与含有Pax 7(1:100)或MyoD(1:300)一抗的200μl封闭缓冲液在4°C孵育过夜。
    4. 在与一抗孵育后,用含有0.5%吐温20的PBS洗涤肌纤维3次,每次5分钟,以除去任何未结合的抗体。
    5. 在室温下与含有适当二抗(1:500)的200μl封闭缓冲液孵育1小时,避光。
    6. 与第二抗体孵育1小时后,每次用含有0.5%吐温20的PBS洗涤5分钟洗涤3次。
    7. 将每根纤维转移到载玻片上并排出剩余的液体。
    8. 盖上盖玻片并在成像前将玻片干燥至少10分钟。用含DAPI的封固剂安装载玻片。
    9. 在荧光显微镜下观察肌纤维并捕获免疫染色肌纤维的图像(图4)。


      图4.在T0,24,48和72小时收获的单个肌纤维培养物的代表性图像。将具有卫星细胞或簇的单个肌纤维固定并用针对Pax7或MyoD的抗体进行免疫染色。红色用于Pax7 <紫色用于MyoD + 卫星细胞。蓝色用于细胞核。比例尺=100μm。

数据分析

在T0处每个肌纤维上的Pax7 + 卫星细胞的平均数量是6-8并且MyoD +

笔记

  1. 用马血清涂布板时避免气泡。
  2. 不要让镊子或剪刀接触EDL肌肉。
  3. 当使用弯曲的塑料滴管转移肌肉纤维时,应沿肌纤维的一端穿入单一的肌纤维,以避免咬住肌肉。
  4. 转移肌纤维时,将喷嘴插入液位以下,以防止肌肉纤维暴露在空气中。
  5. 覆盖玻璃单时应避免气泡。

食谱

  1. 洗涤媒体
    DMEM补充:
    10%胎牛血清
    2%丙酮酸钠
    1%青霉素 - 链霉素
    2 mM EDTA
    通过0.2μm过滤器过滤溶液(可选,去除血清中的任何碎片)
  2. 胶原酶溶液
    DMEM补充0.2%1型胶原酶
  3. 4%多聚甲醛
    1. 将4克多聚甲醛粉末溶于100毫升PBS中。&nbsp;
    2. 将溶液加热至65℃直至多聚甲醛完全溶解
    3. 通过0.2μm过滤器过滤溶液并储存在-20°C
  4. 文化媒体
    DMED补充20%胎牛血清
    1%鸡胚提取物
    1%青霉素 - 链霉素
    通过0.2μm过滤器过滤溶液
  5. 阻塞缓冲区
    PBS补充:
    3%BSA
    0.5%吐温20
    5%山羊血清(在阻断程序之前加入)

致谢

这项工作得到了“GDAS”科技发展项目(批准号2018 GDAS CX-0806)对谢立伟的支持。
作者贡献: LW.X.设计了实验并收集了本研究的资助.LW.X.,SJ.C。,HR.D。和XP.Y.开发并优化了该方法。 SJ.C收集并分析了数据.SJ.C起草了手稿和LW.X.校对手稿。

竞争利益

作者声明不存在财务或研究利益冲突。

道德

C57BL / 6J小鼠在广东微生物研究所(GDIM)的SPF动物设施中以12/12黑暗光照周期饲养,可自由获取食物和水。动物协议由动物保护用途委员会提供。 GDIM(许可#:GT-IACUC201704071)。

参考

  1. 由Bekoff,A。和Betz,W。(1977a)维护的分离的成年大鼠肌肉的特性。在组织培养中。 J Physiol 271(2):537-547。
  2. Bekoff,A。和Betz,WJ(1977b)。从神经支配获得的分散肌纤维的生理特性和分化的成年大鼠肌肉。 J Physiol 271(1):25-40。
  3. Bischoff,R。(1986)。培养中完整肌纤维上肌肉卫星细胞的增殖。 Dev Biol 115(1):129-139。
  4. Collins,CA,Olsen,I.,Zammit,PS,Heslop,L.,Petrie,A.,Partridge,TA and Morgan,JE(2005)。来自成人肌肉卫星细胞生态位的细胞的干细胞功能,自我更新和行为异质性。 细胞 122(2) :289-301。
  5. Danoviz,ME和Yablonka-Reuveni,Z。(2012)。骨骼肌卫星细胞:背景和在原代培养系统中分离和分析的方法。 Methods Mol Biol 798:21-52。
  6. Gallot,YS,Hindi,SM,Mann,AK和Kumar,A。(2016)。隔离,文化和单个肌纤维染色。 生物方案 6(19):e1942。
  7. Lim,CL,Ling,KH和Cheah,PS(2018)。分离,培养和免疫染色单肌纤维:研究卫星细胞行为的改进方法。 J Biol Methods 5(1):e87。
  8. Mauro,A。(1961)。骨骼肌纤维的卫星细胞。 J Biophys Biochem Cytol 9:493-495。
  9. Pasut,A.,Jones,AE和Rudnicki,MA(2013)。个人的隔离和文化myofibers和来自成年骨骼肌的卫星细胞。 J Vis Exp (73):e50074。
  10. Rosenblatt,JD,Lunt,AI,Parry,DJ和Partridge,TA(1995)。养殖卫星生活单肌纤维外植体细胞。 体外细胞生物学动物 31(10):773-779。
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引用:Chen, S., Ding, H., Yao, X. and Xie, L. (2019). Isolation and Culture of Single Myofiber and Immunostaining of Satellite Cells from Adult C57BL/6J Mice. Bio-protocol 9(14): e3313. DOI: 10.21769/BioProtoc.3313.
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