I would like to look at the cell cycle (mouse cells) and distinguish G1/S/G2 phases (not need to be exactly, but I just want to know for sure if my cell population undergo mitosis, and they are quite heterogeneous in terms of mitotic stages).
Does it make sense to check for both cyclins and Cdks at a particular phase? For example, checking Cdk4/Cdk6 and Cyclin D at the same time; or it is enough to conclude by checking for cyclin D only.
I appreciate kind help and explanation from you all. Thank you in advance!
Synchronize the cells by removing serum for 15-18 hours. Then add serum, this will largely synchronize the cells in G1 phase. You can do a time kinetic experiment by collection of samples at various times points say, 0, 3,6,12, 24, 48 hours. Do Propidium Iodide staining and so Dlow cytometry. This will tell you where your cells are being arrested in cell cycle. Now so Cyclin and CDKs of that specific phase for confirmation. Type Fisetin and Gastric cancer in Google, you can read my paper Sabarwal A et.al.
Cdt1 accumulates in G1 and is degraded in S by SCFskp2. Geminin accumulates from S-Phase and is degraded at the onset of anaphase. These genes are also used as probes in the mouse FUCCI system, which is used for live tracing of cell cycle phases.
See: Sakaue-Sawano et. al. 2008
"Visualizing Spatiotemporal Dynamics
of Multicellular Cell-Cycle Progression"
Dear Mai,
two stains will give you a full picture about the cell cycle distribution:
1. BrdU (or better EdU)- labeling + propidium iodide -> clear distinction between G1, S and G2/M phases
2. phospho-Ser10-Histone H3 stain to label mitotic cells -> further distinction between G2 and M cells.
I would label the cells with EdU, split the cells in two parts and run both stains. Both stains can also be done for fluorescence microscopy.
If you just want to determine mitotic index, than ph-H3 test alone would be sufficient.
Good luck!
-CDC25A/B/C - DNA markers are good markers to study the G1/S phase.
-CCND1 (Cyclin D1) is required for G1/S cell cycle transition and can also be used as a G2/M checkpoint marker.
To mark mitotic structures, an antibody against phospho histone 3 (pHH3) could be used.
this link might be helpful: https://ptglab.com/products/featured-products/cell-cycle-and-checkpoint-controls/
Dear all,
Thank you very much for kindly sharing your knowledge and expertise.
I am not really familiar with the cyclins, so I would be very happy to learn more from you all. It is mentioned in one publication in 2003 (Stacey DW, link below) that Cyclin D1 levels are maintained through G1 phase, and then reduced during S phase. However, when the cells enter G2 phase, Cyclin D1 levels rise again. So, I wonder how specific Cyclin D1 is as a G1/S marker. Would it be confusing to define at which phase of the cell cycle our cells are?
I also tried Cdt1/Geminin staining, but the results confuse me again. As referred from the literature, Cdt1 and Geminin are expected to be in the nucleus of mitotic cells. But in my staining, Cdt1 is mostly found in the cytoplasm, while Geminin is localized in both nucleus and cytoplasm. If you have any ideas about Cdt1/Geminin IHC or their localization in the cells, it would be very helpful ...
As far as I remember Cyclin D1 forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition.
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