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-…
Disclosures
The authors have nothing to disclose.
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