Wound Assay to Evaluate the Migratory Capacity of Murine Myoblasts in Co-Culture
Published: April 30, 2023
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 …
Açıklamalar
The authors have nothing to disclose.
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 |
Etiketler
Bu Makaleden Alıntı Yapın
Wound Assay to Evaluate the Migratory Capacity of Murine Myoblasts in Co-Culture. J. Vis. Exp. (Pending Publication), e21287, doi: (2023).