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Cell Biology JoVE (Open Access) Citable · DOI

In Vitro Differentiation of Mature Myofibers for Live Imaging

DOI: 10.3791/55141-v
What you'll learn
  • ✓ Isolate primary myoblasts from neonatal mouse hind-limbs
  • ✓ Differentiate myoblasts into mature, multinucleated myofibers in vitro
  • ✓ Perform immunostaining and live imaging of developing myofiber cultures
  • ✓ Identify developmental stages from myoblast to mature myofiber
Protocol

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

Difficulty
advanced
Total time
~3-4 days (dissection ~30 min, isolation ~2 hrs, differentiation 2-3 days, imaging variable)
Model organism
Mouse (neonatal hind-limb)
Biosafety
BSL-1

Steps

1
Dissect neonatal mouse hind-limbs

Isolate hind-limbs from neonatal mice using sterile technique. Prepare tissue for myoblast extraction.

▶ 00:46
2
Isolate myoblasts from dissected tissue

Enzymatically digest muscle tissue and isolate myoblast cells using mechanical trituration and differential plating methods.

▶ 02:09
3
Differentiate myoblasts and perform immunostaining

Culture myoblasts under differentiation conditions and apply immunostaining protocols to visualize myofiber maturation and developmental markers.

▶ 04:09
4
Image and characterize developmental myofiber stages

Acquire live or fixed imaging data showing progression from myoblasts through multinucleated mature myofibers during culture.

▶ 06:10
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