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)

1. 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.
2. Warm 1x PBS and C2C12 medium by placing them in a 37 °C water bath.
3. Remove supernatant medium from the C2C12 chamber well and wash the cells once with 1x PBS. Remove the 1x PBS and immediately scratch the cells wi....

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 pe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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