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Cell cycle analysis with BrdU and propidium iodide

HZ Hui Zhao
BT Bernard Thienpont
DL Diether Lambrechts

Last updated date: Jul 26, 2022 Views: 571 Forks: 0

original An abbreviated version of this protocol was published in eLife in Aug, 2014
Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks
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  1. Seed 300,000 cells/well in 6-well plates and treat with 26uM olaparib, 300nM mitomycin C, 30nM camptothecin or placebo.
  2. After 24 hours, incubate cells with BrdU at a final concentration of 10uM for 90 minutes.
  3. Trypsinize cells for 5 min.
  4. Add 2mL medium to stop trypsinization
  5. Centrifuge @400g, 5 min, 4°C
  6. Resuspend the pellet in 500 uL ice-cold PBS.
  7. Slowly add 2mL 100% ice-cold ethanol is while mixing the resuspension
  8. Incubate for 5 minutes at room temperature to allow fixation.
  9. Centrifuge @400g, 5 min
  10. Resuspend in 2mL PBS for washing
  11. Centrifuge @400g, 5 min, 4°C
  12. Resuspend in 2mL PBS for washing
  13. Centrifuge @400g, 5 min, 4°C
  14. Resuspend in 2M HCI/0.05% Tween20 in PBS for 30 minutes to denature DNA
  15. Centrifuge @400g, 5 min, 4°C
  16. Resuspend in 0.5% normal goat serum/0.05% Tween20 in PBS, incubate for 10 minutes at room temperature.
  17. Add FITC-conjugated anti-BrdU antibody diluted 1:10 in 0.5% NGS/0.05% Tween20 in PBS at room temperature.
  18. Centrifuge @400g, 5 min, 4°C
  19. Wash away antibody by resuspending the pellet with 0.05% Tween20 in PBS
  20. Centrifuge @400g, 5 min, 4°C
  21. resuspend pellet in 25 ug/mL propidium iodide containing 0.1 mg/mL RNase A.
  22. Measure fluorescence on a BD Biosciences FACSVerse flow cytometer
  23. Model the cell cycle data using FlowJo software. The fraction of cells in S-phase, G2/M and G1 are determined as described by Watson et al. (1987).
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Zhao, H, Thienpont, B and Lambrechts, D(2022). Cell cycle analysis with BrdU and propidium iodide. Bio-protocol Preprint. bio-protocol.org/prep1821.
  2. Zhao, H., Thienpont, B., Yesilyurt, B. T., Moisse, M., Reumers, J., Coenegrachts, L., Sagaert, X., Schrauwen, S., Smeets, D., Matthijs, G., Aerts, S., Cools, J., Metcalf, A., Spurdle, A., Amant, F. and Lambrechts, D.(2014). Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks. eLife. DOI: 10.7554/eLife.02725

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