Cell Cycle

Monitoring of cell cycle

1. Using fixed cells and propidium iodide (PI) to measure the DNA content and monitor cell cycle and its regulation.

Other DNA dyes:

UV excitation: Hoechst, DAPI

488 excitation: PI, 7AAD

633 excitation: TO-PRO-3

2. DNA staining of live cells using Hoechst 33342 (goes to all cells) and DRAQ7 (only dead cells).

3. Using bromodeoxyuridine (BrdU) which is thymidine analog taken up by cycling cells – S phase easy to separate from G1 and G2/M, more accurate results

on the basis of Peter O'Toole, Cytometry Course 2019, York

Proliferation tracking

The flow cytometer can measure proliferation of cells labeled with a cell membrane fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE) or other freqently used dyes like Cell Proliferation Dye eFluor™ 450 or 670, CellTrace™ or BD Horizon™ Violet Proliferation Dye 450 (VPD450). When the cells are activated, they start to devide. During mitosis, half of the original dye is passed on to each daughter cell. By measuring the reduction of the fluorescence signal, one can calculate the number of cells in each of the proliferation rounds.