A Technique to Isolate Satellite Cells From Rat Head Muscle Tissue

Abstract

Source: Carvajal Monroy, P. L., et al. Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles. J. Vis. Exp. (2015).

This video demonstrates the isolation of satellite cells from rat head branchiomeric muscle tissues through tissue dissection, enzymatic digestion, and subsequent mechanical and centrifugal purification. The cells are then seeded on extracellular matrix-coated wells for differentiation into myoblasts, and eventually into myotubes and mature muscle fibers.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Extracellular Matrix Gel Spots Perform the following steps one day before the isolation: Thaw an aliquot of extracellular matrix gel (100 μl) at 4 °C for at least 1.5 hr. Dilute 1:10 in Dulbecco's modified Eagle's medium; with 4,500 mg/L glucose, 4 mM L-glutamine, and 110 mg/ml sodium pyruvate (DMEM). Keep the extrac…

Representative Results

Figure 1: Extracellular matrix gel spots in a chamber slide. (A) For easy manipulation, place the 8-well chamber slide into a 100 mm Petri dish. Pipet 10 μl extracellular matrix gel in each chamber and put it on a cold surface (7 min). (B) Chamber slide after the excess extracellular matrix gel is removed. <p class="jove_content" fo:keep-together.within-…

Materials

Hypodermic Needle 25 G  0.5 x 25 m	BD Microlance	300400
Dissecting scissors	Braun	BC154R
Micro forceps straight	Braun	BD330R
Surgical Scalpel Blade No. 15	Swann-Morton	0205
Alcohol 70%	Denteck	2,010,005
Permanox Slide, 8 Chamber	Thermo Scientific	177445
6 well cell culture plate	Greiner bio-one	657160
Cell Culture Dishes (100 x 20 mm)	Greiner bio-one	664160
15 ml sterile conical centrifuge tube	BD Biosciences	352097
50 ml sterile conical centrifuge tube	BD Biosciences	352098
Cell strainer (40 μm)	Gibco	431750
10 ml serological pipette	Greiner bio-one	607180
20 µl FT20	Greiner bio-one	774288
Matrigel, Phenol-Red Free	BD Biosciences	356237	10 ml
Pronase	Calbiochem	53702	10 KU
Phosphate Buffered Saline	Gibco	14190-144	500 ml
Dulbecco's Modified Eagle Medium, high glucose, GlutaMAX Supplement, pyruvate	Gibco	10569-010	500 ml
Fetal Bovine Serum	Fisher Scientific	3600511	500 ml
Horse Serum	Gibco	26050088	500 ml
Penicillin-Streptomycin (10,000 U/ ml)	Gibco	15140-122	100 ml
Chicken Embryo Extract	MP Biomedicals	2850145	20 ml