Sep 29, 2025
Cell Cycle Analysis by Flow Cytometry
- Maria Jose Perez J.1
- 1Institut Imagine
- Team Deleidi
Protocol Citation: Maria Jose Perez J. 2025. Cell Cycle Analysis by Flow Cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3176el25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 25, 2025
Last Modified: September 29, 2025
Protocol Integer ID: 228168
Keywords: flow cytometry cell cycle analysis
Funders Acknowledgements:
ASAP
Abstract
Cell Cycle Analysis by Flow Cytometry
Troubleshooting
Dissociate iPSC-derived cells using Accutase and resuspend cells in PBS.
Stain cells with Hoechst 33342 at 2.5 µg/mL for 30 minutes at 37°C.
Wash cells with PBS
Incubate cells with Zombie NIR viability dye at 1:1000 dilution in PBS to exclude dead cells
Wash cells with PBS.
Resuspend cells in PBS containing 2% fetal bovine serum (FBS).
Transfer cells into 5 mL round-bottom tubes for acquisition
Perform flow cytometry on a Sony ID7000‱ spectral cell analyzer (Sony Biotechnology, USA) at a maximum event rate of 200 events per second.
Analyze acquired data using the cell cycle analysis module in FlowJo‱ software (BD Biosciences).
Apply the Watson (Pragmatic) model for cell cycle quantification.
Distinguish G1, S, and G2/M phases based on Hoechst signal intensity (G1 in red, S in light gray, G2/M in blue).
Visualize model fitting by overlaying the model sum (dark gray) and actual signal distribution (black line).
Constrain parameters by setting Peak 1 to "n" and constraining Peak 2 to match the coefficient of variation (CV) of the G1 peak.
Select the final model based on achieving a low root mean square deviation (RMSD) value.