I have been using Edu/Brdu + PI to analyze cell cycle status. This method can well distinguish cells in G0/G1, S, G2/S with zero/minimal overlapping.
After treatment, cells in G0/G1 phase could be arrested in G1 or still proliferating. How to discriminate them? I am wondering Is there a good marker(s) for cell cycle arrest at G1 phase (like phosphatidylserine exposure which could be picked up by AnnV staining for apoptosis).
How about dephosphorylation of Rb protein? Thanks
You could try adding BrdU and then inmunostaing with Ab-BrdU. If the cells are actually blocked in G1 they should not incorporate BrdU, and show no or low fluorescence. Use as a control for BrdU fluorescence an exponentially growing culture.
Please check the following lines from Abcam, if suits to your work:
The Cell Cycle western blot cocktail (ab136810) is designed to provide a rapid assessment of cell cycle distribution based on molecular markers for the G1/S and M phases of the cell cycle. This is a mixture of three specific rabbit monoclonal primary antibodies targeting phospho-cdk2 Tyr15, phospho Histone H3 Ser10 and beta-actin. Cyclin dependent kinase 2 (Cdk2) is maintained in an inactive state in G1/S by inhibitory phosphorylation on Tyr15. Histone H3 is phosphorylated at Ser10 when chromosomes condense during mitosis. Hence, elevated phosphocdk2 Tyr15 indicates G1/S arrested cells and elevated phospho Histone H3 Ser10 indicates M-phase arrested cells when compared to asynchronous cycling control cells. An anti- Actin antibody is included as a loading control. These three readouts are easily resolved by western blot given their different molecular weights.
Dr. Mukherjee
for traking cell cycle phases you can use FUCCI . fragments of cdt1(G1 marker) and geminin(G2 to M marker) were tagged to RFP and GFP respectively. when cells are in G1 they express cdt1-RFP hence cells show red flurorescence. the same cell when come to S phase start degrading cdt1-RFP and start expressing geminin-GFP leads to both red and green (composite image become yellow) fluorecense. from roughly mid of the S phase presence of only geminin make cells to show only RFP but not GFP gives indication of mid S or G2 or M phase.
these constructs are available commertially.
http://www.amalgaam.co.jp/products/advanced/fucci.html
http://www.lifetechnologies.com/in/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-cycle/live-cell-imaging-of-cell-cycle-and-division.html
recently this system is optimised for mice and zebrafish too.
If I well understand, you want to discriminate between cells that are in G1 and still cycling and cells that are arrested at the restriction point or going back to G0. Am I right? Before answering, it will be important to know whether you want to get a single cell resolution (FACS analysis) or if you are looking from a population point of you. What type of equipment could you access? FACS, Cell Scan Array? What type of cells? Treatment induces differentiation or arrest and cell death? Answering these questions will orient the answer. As a good start I well recommend intracellular staining with cycline D, E and CKI p16, p21, p27.
Thanks for your answer, Dominique,
Yes, you got the question right.
Equipment: flow cytometer and FACS, analyze a population of cells (to get %) not on single cell level.
Cell type: Leukemia cells
Treatment will induce both arrest at G1 and apoptosis.
I am not looking for the causes/dynamics of cell cycle arrest (cyclin changes, p21...), but the results/markers for it (e.g. beta-galactosidase as a marker for senescence). After Edu+PI staining, we can see cells pile up in G1/G0. This could be due to cell cycle arrest at G1 or exhaustion of S/G2/M phases cells due to cell death (G1 cells might be still cycling)or other reasons. Therefore, a marker for G1 arrest will be an easy and clear answer.
I think that you are close to the question I was investigating a few years ago. We spent a significant amount of time to do time course FACS analysis to be convince by a mechanism but conviction is not demonstration (G1 arrest was preventing cells to go under apoptosis). Today, I would recommend to perform timelapse microscopy, you are going to have your answer in a overnight experiments.
I believe there is no simple marker for G1 arrested cells. The approach most commonly used is to analyse multiple markers (e.g. in WB) in a time-course experiment. It seems that you don't want or can not use time-course analysis? In G1 arrest (but not in cycling G1 cells), p53/p21/p27/p16 levels (often) go up, but phospho-Rb or cyclin protein levels are not good markers since they change during normal cycle. As in cancer cells p53/p21 etc pathways can be disturbed, multiple markers are usually analysed.
Quote: "KI-67 is commonly used as a marker of proliferation, identifying tumor cells in the non-G0 state. Loddo et al (British Journal of Cancer 2009; 100: 959–970.) recently showed that KI-67 is a suboptimal marker of proliferation, since many tumor cells that express KI-67 undergo G1 arrest, exit the cell cycle or undergo apoptosis rather than complete the cell cycle." Here I am trying to identify G1-arrested cells.
A good point though. Thanks
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