Hello fellow researchers,
In one of my experiments, I need to identify cells in G0 phase in my primary cell culture. It will be very helpful if anyone have any experience with such experiment and can suggest any marker based or flow based assay.
Thanks in advance
Hello,
Try looking up the following
BrdU (+/- 7-AAD and PI)
Ki67
BrdU is incorporated in new DNA while Ki67 stains proteins that are required for proliferation. Therefore, cells negative for BrdU or negative for Ki67 are G0 cells as they do not synthesize new DNA and do not express those proliferation proteins, respectively. BrdU is better for cynetics. If you add BrdU to the cell culture for 24h, any negative cell is definitely in G0 and has been in G0 during those 24h.
There are plenty of protocols on the internet on various sites of various manufacturers.
Good luck!
You can do Propidium Iodide (PI) staining and go for flow cytometry to know the cell cycle phase of the cells.
FACS analysis after Propidium Iodide staining is fine if you are not concerned about whether the cells are in G1 phase as well, because both phases will have identical DNA content.
The classic way of identifying quiescent cells in a population is by label retention. A label is added to the media like 5′-bromo-2′-deoxyuridine (BrdU) or tritiated thymidine and allowed for incorporation into all the cells. Then the label is washed off and fresh media is added. Dividing cells will lose the label compared to quiescent cells which more or less retain the label without loss because of their low metabolic activity.
Alternatievly, If you want to sort the number of cells that are in G0, you can tag quiescence signatures present only in G0 cells. These are downregulated genes for replication (MCM4, PCNA) mitochondrial activity (CYCS) cell cycle (CCNB1) and chromatin assembly (HAT1) and upregulated genes like FOXO3 and several CDK inhibitors. There is a whole list of them that can be used as markers found in this article (doi: 10.1038/nrm3591).
Dear Rahul Kumar by using DNA dye you will be able to separate G0/G1, S and M phase but if you add RNA dye as well, you will be able to separate G0 and G1 by flowcytometry. Attaching you an article showing protocol and gating for the same
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