Protocol to assess myogenic effects of 3T3-L1 and primary murine fibro-adipogenic progenitors on C2C12 myoblast differentiation

Fibro-adipogenic progenitors (FAPs) are key regulators of skeletal muscle regeneration and influence myogenic differentiation. Here, we present a protocol for the isolation of primary FAPs from injured murine skeletal muscle and the co-culture of C2C12 myoblasts with either 3T3-L1 or primary FAPs. We describe steps for muscle injury, harvest, and digestion followed by FAP isolation. We then detail procedures for co-culture and for assessing myogenic differentiation using immunofluorescence imaging, enabling direct comparison of stromal influences on myoblast differentiation. For complete details on the use and execution of this protocol, please refer to Norris et al. 1 • Isolate primary fibro-adipogenic progenitors from injured skeletal muscle • Co-culture C2C12 myoblasts with either 3T3-L1 preadipocytes or primary FAPs • Compare effects of 3T3-L1 and primary FAP population on myoblast differentiation • Quantify myoblast differentiation using immunofluorescence imaging Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Fibro-adipogenic progenitors (FAPs) are key regulators of skeletal muscle regeneration and influence myogenic differentiation. Here, we present a protocol for the isolation of primary FAPs from injured murine skeletal muscle and the co-culture of C2C12 myoblasts with either 3T3-L1 or primary FAPs. We describe steps for muscle injury, harvest, and digestion followed by FAP isolation. We then detail procedures for co-culture and for assessing myogenic differentiation using immunofluorescence imaging, enabling direct comparison of stromal influences on myoblast differentiation.