Hello everyone,
When determining cells in different stages of the cell cycle, cells are stain for Brdu and Propidium Iodide. I understand how PI can be used to discriminate between G1 and S/G2 cells but I'm not sure how Brdu is used to distinguish between S and G2 cells. Why is it that Brdu staining drops after S phase?
Hi Rafael. I assume that you incubate cells with BrdU for ~2hrs and immediately fix them for immunostaining. Actually the FACS plot is intended to show cell cycle state of cells within the BrdU incubation time. So, the cells entering S-phase will incorporate BrdU nucleotides into newly synthesized DNA within that 2hr period and will stain positive for BrdU. But if cells are already in G2/M or G1 within that 2hrs of BrdU incubation, they won’t have BrdU incorporated and will show weaker or no signal. That separates S-phase cells from G2/M. And the higher intensity of PI signal separates G2/M from G1 cells. Hope my explanation makes sense.
Dear Rafael!
You can also see the information on the website
https://www.abcam.com/protocols/flow-cytometric-analysis-of-cell-cycle-with-propidium-iodide-dna-staining
Hi Rafael,
to answer your specific question:
1. BrdU is incorporated as a thymidine analog into nuclear DNA during DNA synthesis
2. DNA is synthesized only during S phase
3. when you pulse with BrdU, you are only incorporating into those cells that are in S phase during the pulse. sort of like a snap shot.
4. Cells that are in G1, G2, and Mitosis will not incorporate and thus appear lower on the FITC axis. not because the signal has dropped, but because of the snap shot effect
Kind regards, Lora
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