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. 2019 Jul 20;9(14):e3313.
doi: 10.21769/BioProtoc.3313.

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

Shujie Chen  1 , Hongrong Ding  1 , Xiangping Yao  1   2 , Liwei Xie  1   2
Affiliations

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

Shujie Chen et al. Bio Protoc. .

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: Immunofluorescence staining; Muscle satellite cells; Myofiber isolation and culture.

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Conflict of interest statement

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

Figures

Figure 1.
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.
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].
Figure 2.
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.
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.
Figure 3.
Figure 3.. Images of isolated single myofiber after cultured for 0/24/48/72 h
. Scale bars = 100 μm.
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.
See this image and copyright information in PMC

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