Assessing Muscle Contraction with Neuromuscular Blocker Treatment

Abstract

Source: Lin, C. U., et al., In Vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells. J. Vis. Exp. (2020)

This video demonstrates assessing the muscle using cultured motor neurons, Schwann cells, and myotubes, followed by treatment with a neuromuscular blocker to inhibit contraction. Color-coded time-motion graphics show a color-coded time-motion graphic. Red indicates the fastest movement, and blue indicates the slowest.

Protocol

1. Muscle contraction and curare treatment

1. To trigger the myotube contraction, add 25 mM calcium chloride (CaCl₂) to the culture medium. The movement of myotubes can be observed in 1‒2 min.
2. Place the 6-well plate on the stage of an inverted microscope. Record a movie of myotube contraction by live cell microscopy.
3. To stop the myotube contraction, add curare to the culture medium (300 ng/mL). Then, record the movie as step 1.2.
4. Open the movie file with motion vector analysis software, then click the Motion Analysis button to analyze the movie.
   NOTE: The myotubes contraction is shown as time-motion graphic. The movies are color-coded to show the movement speed of myotubes. The color-coded sparkly signals indicate the movements of myotubes where the red color shows the fastest movement speed and blue color shows the slowest movement speed.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Medium, growth factors and reagents	Item	Brand	Cat. Number
	B27, Gibco	Gibco	12587-001
	BDNF	R & D Systems	248-BD
	ECM, Matrigel (growth factor reduced)	Corning	356230
	GDNF	R & D Systems	212-GD
	Ions coater	JEOL	JEC3000FC
	Live cell microscopy	Nikon	Eclipse Ti microscope
	Motion vector analysis software	Sony	SI8000
	N2, Gibco	Gibco	17502-048
	Neurobasal medium	Gibco	21103-049
	NT3	R & D Systems	267-N3