Related Experiment Video
Updated: Jul 13, 2025
10:20
Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells
Published on: March 24, 2023
1.6K
A flow-cytometry-based pipeline for the rapid quantification of C2C12 cell differentiation
Bianca Parisi1, Maxime Sünnen1, Rohan Chippalkatti1
1Cancer Cell Biology and Drug Discovery Group, Department of Life Sciences and Medicine, University of Luxembourg, 4362 Esch-sur-Alzette, Luxembourg.
STAR Protocols
|October 11, 2023
Summary
This study introduces a flow cytometry method to measure C2C12 cell differentiation using myosin heavy-chain markers. The FlowFate R script automates this analysis, enabling studies on gene effects in differentiation.
Area of Science:
- Cell Biology
- Biotechnology
- Molecular Biology
Background:
- The C2C12 cell line is a widely used in vitro model for studying skeletal muscle differentiation.
- Quantifying cell differentiation accurately is crucial for understanding muscle development and disease.
- Existing methods for assessing differentiation can be labor-intensive and subjective.
Purpose of the Study:
- To develop and validate a flow cytometry-based pipeline for quantitative assessment of C2C12 cell differentiation.
- To introduce an automated analysis tool, the FlowFate R script, for processing flow cytometry data.
- To demonstrate the utility of the pipeline and script in evaluating the dose-dependent effects of specific genes on differentiation.
Main Methods:
- Established a detailed protocol for C2C12 cell culture, including seeding, transfection, drug treatment, and differentiation induction.
- Utilized myosin heavy-chain expression as a marker for differentiated myotubes.
- Employed flow cytometry for high-throughput acquisition of cell differentiation data and developed the FlowFate R script for automated analysis.
Main Results:
- Successfully developed a robust flow cytometry pipeline capable of quantifying C2C12 cell differentiation based on myosin heavy-chain expression.
- The FlowFate R script provides automated and reproducible analysis of flow cytometry data.
- Demonstrated the ability to assess the dose-dependent impact of GFP-tagged genes on C2C12 cell differentiation using the developed pipeline and script.
Conclusions:
- The presented flow cytometry pipeline offers a quantitative and efficient method for studying C2C12 cell differentiation.
- The FlowFate R script significantly streamlines the analysis of differentiation data, facilitating high-throughput screening and mechanistic studies.
- This approach provides a valuable tool for researchers investigating the molecular mechanisms regulating skeletal muscle differentiation.
