成年C57BL/6J小鼠单个肌纤维的培养分离和卫星细胞的免疫染色

Shujie Chen; Hongrong Ding; Xiangping Yao; Liwei Xie

doi:10.21769/BioProtoc.3313

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Peer-reviewed

Isolation and Culture of Single Myofiber and Immunostaining of Satellite Cells from Adult C57BL/6J Mice

成年C57BL/6J小鼠单个肌纤维的培养分离和卫星细胞的免疫染色

(*contributed equally to this work) 发布: 2019年07月20日第9卷第14期 DOI: 10.21769/BioProtoc.3313 浏览次数: 8248

评审: Zijian ZhangAnonymous reviewer(s)

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Cover of The Journal of Clinical Investigation, featuring study using the protocol.

Jun 2018

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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

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

Equipment

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

Procedure

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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.