biostudies-literatureUnknown4(4)Parisi BThe C2C12 cell line represents a simple in vitro model for cell differentiation. Here, we present a flow-cytometry-based pipeline to quantitate C2C12 cell differentiation based on myosin heavy-chain marker expression. We describe steps for cell seeding, transfection, drug treatment, differentiation, and labeling. We then detail procedures for flow cytometry acquisition and introduce the R script FlowFate for automated analysis, including the study of dose-dependent effects of GFP-tagged genes on differentiation. For complete details on the use and execution of this protocol, please refer to Chippalkatti et al. (2023).¹.STAR protocols102637https://www.ebi.ac.uk/biostudies/studies/S-EPMC10568640biostudies-literatureA flow-cytometry-based pipeline for the rapid quantification of C2C12 cell differentiation.PMC10568640Parisi BSunnen MAbankwa DKChippalkatti RfalseA flow-cytometry-based pipeline for the rapid quantification of C2C12 cell differentiation.The C2C12 cell line represents a simple in vitro model for cell differentiation. Here, we present a flow-cytometry-based pipeline to quantitate C2C12 cell differentiation based on myosin heavy-chain marker expression. We describe steps for cell seeding, transfection, drug treatment, differentiation, and labeling. We then detail procedures for flow cytometry acquisition and introduce the R script FlowFate for automated analysis, including the study of dose-dependent effects of GFP-tagged genes on differentiation. For complete details on the use and execution of this protocol, please refer to Chippalkatti et al. (2023).¹.2023-01-01T00:00:00Z2023 Oct2025-04-22T11:03:18.84Z2025-04-05T23:47:31.556ZS-EPMC105686403781976210.1016/j.xpro.2023.102637