Wound Assay to Evaluate the Migratory Capacity of Murine Myoblasts in Co-Culture

Abstract

Source: Toubat, O. et al., Modeling Paracrine Noncanonical Wnt Signaling In Vitro. J. Vis. Exp. (2021).

This video describes a non-contact co-culture wound healing assay to evaluate the migratory capacity of murine myoblasts. This helps to understand signal-sending and signal-receiving components of the paracrine non-canonical Wnt signaling interactions in vitro.

Protocol

1. Wound assay: (Figure 1 outlines the schematic model of the assay) Allow the O9-1 cell inserts and C2C12 cell chamber wells to adhere and proliferate in the incubator until both cells are at ~70-80% confluency before proceeding with this portion of the protocol. If cells grow >90% confluent, do not proceed with the scratch assay, as cells will merely detach from the well. Warm 1x PBS and C2C12 medium by placing them in a 37 °C water bath. …

Representative Results

Figure 1: Schematic model of the assay. Step 1 includes the in vitro expansion of C2C12 myoblasts and NCCs using STO feeder cells. Step 2 involves the plating of NCCs and C2C12 cells in the coculture system. Step 3 includes the wound assay performed in underlying C2C12 cells to evaluate cellular migratory capacity. Step 4 involves immunostaining for phalloidin to evaluate …

Materials

C2C12 murine myoblast cell line	ATCC	CRL-1772
Chamber Slide System, 4-well	ThermoFisher Scientific	154526
Dulbecco’s Modified Eagle’s Medium (DMEM), high glucose (4.5 g/L), Lglutamine (2 mM)	Corning	10-017-CV	Stored at 4 °C
Graduated and sterile pipette tips, 10 µL	USA Scientific	1111-3810
O9-1 neural crest cell line	Millipore Sigma	SCC049
Phosphate-buffer saline (PBS), 1x, without calcium and magnesium, pH 7.4	Corning	21-040-CV	Stored at 4 °C
Falcon permeable support for 24-well plate with 0.4 µM transparent PET membrane	Corning	353095
Fetal bovine serum	Fisher Scientific	W3381E	Stored in 50 mL aliquots at -20 °C
Zeiss inverted Axio Vert.A1 light microscope	Carl Zeiss AG
Zen lite 2012 microscopy software	Carl Zeiss AG		Imaging software