Cell cycle analysis by PI is like to take a snapshot of a busy street. You can see how many cars are in the parking lane, in the slow lane and in the fast lane. However you can not be sure that accumulation of cars in both slow and fast lanes means many cars passing by or a complete traffic jam. Similarly with the call cycle analysis you can see cells accumulating in S-phase and it can mean two things: cells proliferate more or they are arrested (e.g. because of DNA damage) in the middle of duplicating their DNA. You need more information to solve this problem. Easiest thing is to look at the outcome: Is that supermarket parking lot at the and of my street filling up with cars? Do I have more cells in the group where I can see more cells in S-phase? There is another way how to discriminate between proliferation and cell cycle arrest. For a limited time you feed your cells with a labeled nucleotide (e.g. bromodeoxyuridine) and than you use an antibody with a fluorescent marker to identify cells incorporating BrdU. If they are positive, you can be sure that they synthetize DNA (and incorporate BrdU). If they are (in double staining with PI) in S-phase and at the same time BrdU negative, that means cell cycle arrest.

Dear Bortolotto,

After u run the flow cytometry, u have to lock to the percentage of each (I mean G1, S, and G2/M phases), and then u compare these percentages with treated group, so u can realize your drug cause cell cycle arrest at each phase, In addition, if your drug could induce apoptosis u will see small percentage before G1 phase. it is so easy to interpret the flow chart. Just start and do it and u feel it is so easy.

Regards,

Bashir 

In the page 4 of attached paper, you will be able to see an example about cell cycle arrest. Untreated cells remain in a balanced distribution of the cell cycle as cells vehicle (DMSO), while cells treated with (1) change their distribution and accumulate in G2 / M phase. This means that there was cell cycle arrest in the G2 / M phase because (1). Hope that helps.

Cell cycle analysis by PI is like to take a snapshot of a busy street. You can see how many cars are in the parking lane, in the slow lane and in the fast lane. However you can not be sure that accumulation of cars in both slow and fast lanes means many cars passing by or a complete traffic jam. Similarly with the call cycle analysis you can see cells accumulating in S-phase and it can mean two things: cells proliferate more or they are arrested (e.g. because of DNA damage) in the middle of duplicating their DNA. You need more information to solve this problem. Easiest thing is to look at the outcome: Is that supermarket parking lot at the and of my street filling up with cars? Do I have more cells in the group where I can see more cells in S-phase? There is another way how to discriminate between proliferation and cell cycle arrest. For a limited time you feed your cells with a labeled nucleotide (e.g. bromodeoxyuridine) and than you use an antibody with a fluorescent marker to identify cells incorporating BrdU. If they are positive, you can be sure that they synthetize DNA (and incorporate BrdU). If they are (in double staining with PI) in S-phase and at the same time BrdU negative, that means cell cycle arrest.

Thanks, Dr. Yousef and Medina. Your help was really important to me! 

And it was very well explained, Dr. Andrysik. These information are fundamental for my work. Thank you very much!

You are welcome. And BTW, pricing of the BD kit is a total rip-off.

If you suspect a delay of G1 and/or S phase progression or arrest in G1/S then you might also want to use Nocadazole to arrest cells in G2. If cells exhibit delayed progression in G1 or S then the percentage of cells in G2/M will be lower.

I agree that double labeling cells with BrdU or EdU is best for cell cycle analysis. Be advised to keep the labeling period confined to 30 min however. In addition, if you want to discriminate G2 from M phase you will need an additional marker such as H3-P (Ser-10). 

As alternative to BrdU or EdU you might want to look at FUCCI, which can be used for flow as well, but is might be more expensive. On the other hand it is fast. 

Regarding the software for analysis, in the end it depends on what your institution has available. Personally I am not a big fan of ModfFit, preferring FlowJo. It helps a lot to use sample data provided in order to get "feel" of the software and learn gating. Also, as always, read papers and pay attention to the gating strategy they used. If you have an administrator for the flow facility, he or she might be the person to ask for assistance. 

I've read many papers about cell cycle arrest and most of them points to G1 and G2 arrest, ranging from one to another (obviously depending on drug and treatments) Few articles on S phase arrest.

My Annexin V-FITC/PI results show apoptosis as PCR array and other experiments do. I've been doing cell cycle tests and its results point to not much significant, but believable cell accumulation on G1 in detriment of the S and G2/M phases. So, is this a cycle arrest in G1? 

Cell cycle arrest in each phase has its specific interpretation, but the one with G2/M arrest mean the best one that inhibit mitosis and proliferation of cancer cell. Tq

as we can see on the histogram there is a little shift to the right in KO cells in comparaison to control (red ) == more fluorescence in ko . how can i interpret it ?

more DNA in ko

thank you

it is quite simple: Cells accumulate in G1 means that your drug has arrested it at this phase, BTW most of the drugs do this eefect and it could not be used as indicator on the potency of this drugs and anticancer agent for example. the best drug should arrest cells at G2/M phase, which mean that it interfere with the mitotic division.