For cell cycle analysis, we normally do not use live cells. If this is not necessary for your protocol I can advise you to fix them in 70% EtOH for at least two hours at -20 degrees. You can then omit the triton X-100 from the staining solution since the EtOH also permeabilizes the cells. 

You should fix the cells with 70% ethanol and during this procedure, you should pipet the cells using tips as much as possible to make the cells separated as a single cell.

I agree with all of the above. Fixing with 70% Ethanol at 4 degrees is very important for your cell cycle analysis. I use a similar protocol regarding the staining and it works perfectly. It is sometimes very difficult to distinguish the three peaks (G1, S and G2/M) from each other due to overlapping. I suggest after you have completed all your runs, save your data in an FCS format and analyse it using the De Novo FCS Express software 6.  The software detects the peaks and gives a percentage of the cellular population occupying that cycle. 

I think you should fix the cells with absolute 20-50 ul methanol/ethanol (quickly) followed by a 5-10 minutes incubation at -20 degree C and then rehydration with 500 ul PBS in 4 degrees for 30 minutes. You may then carry on with RNase treatment for better results.

Moreover it is important that at every step, especially before fixing the pellet should be well dissolved in residual PBS resulting in a single cell suspension so that more and more cells are exposed to the fixative.

Staining for cell cycle analysis by DNA content with using PI, RNAse and Triton X-100 is a routine method and you will get good results very soon at least for intact cells. Method is really simple and has many modification in different labs but unfortunately some of the using protocols have lost "well known" or "unimportant" details leading to confusing results.

The first steps below are just fixation but they are important for followed results.

1. Wash cells 2 times by centrifugation (e.g. 200 x g, 5 min, 4°C) in protein-free buffer, such as Phosphate Buffered Saline without Ca+2 or Mg+2 (PBS).

2. Resuspend at 1-2x106 cells in 1 ml ice cold buffer (Cell number will effect staining quality! It is beeter to use pre-coated or silanized polypropylene tubes to minimize sticking. You may pre-coat tubes overnight with 2% Bovine Serum Albumin in PBS).

3. Vortex gently, slowly adding the cell suspension dropwise to 9 ml of 70% ethanol in a 15 ml polypropylene centrifuge tube. OR: Vortex gently, slowly adding the cell suspension dropwise to an equal volume of cold absolute ethanol. (you may ferivy of cell preparation is better, t.e. less or more clumps, with using a microscope).

4. Store at 4°C to - 40°C for at least 2 hours (12 - 24 hours is best). In principle, cells could be stored weeks without changing of the results.

5. Centrifuge cells for 10 minutes, aspirate the supernatants being careful not to lose the pellet. Note that ethanol-fixed cells require a bit higher centrifugal speeds to pellet compared to unfixed cells since they become more buoyant upon fixation. 

6. Resuspend pellet in 3 ml cold PBS and transfer to polystyrene tubes for staining. You may use additional nylon filter caps to remove possible clumps.

Now we can start staining with Propidium Iodide (PI).

7. Wash cells at least once with COLD PBS. Cells may form a diffuse ring-shaped pellet, so centrifuge longer ( e.g. 200 x g, 10 min, 4°C).

8. Resuspend cells in 300 - 500 µl PI/Triton X-100 (in agreement with answer  above you can exclude triton X-100 from the staining solution after fixation but it does not destroy the results) the staining solution: to 10 ml of 0.1 % (v/v) Triton X-100 in PBS add 2 mg DNAse-free RNAse A and 0.40 ml of 500 µg/ml PI. It would be better if it would be prepared freshly. However, a stock solution of PI itself, made by dissolving 1 mg PI in 2 ml water, can be stored several months at 0° to 4°C. (if the RNAse is not DNAse-free, boil a solution of 2 mg RNAse A in 1 ml water for 5 min. Aliquot and store at -20°C).

9. Incubate 37°C for 15 minutes or for 30 min at 20°C (some cells e.g. fibroblasts may need longer incubation period to stain properly).

10. Transfer tubes to ice or store at 4°C (protected from light).

11. Analyse samples by flow cytometry ideally within 48 hours (in principle, the sample might last longer, at the same time it may require additional filtration to remove arising cell clumps). You do not need to wash cells again and can analyse cells directly in PI/RNase solution. ls). Save at least 10,000 single cell (you have spent so long time for the experiment and cell preparation, why dont you spend 1-2 minutes longer for ther analysis).

Thanks so much for everyone. I did use 70% ethanol to fix the cells and put the cells in -20 degree for several days. I want to ask some questions. 1. when we gives the cells to the centrifugation, is it ok with the speed of 1500rpm? is it so strong that makes the cells rupture? 2. after centrifugation, we dissolve the cells with PBS, should we pipette the cells strongly to make the cells separated? 3. When we fix the cells, should we add the cell solution into ethanol or we add ethanol into the cell solution? Does this influence the results?

For the cells to get fixed in 70% ethanol it takes not more than 30 minutes I guess. And although it is preferable to acquire the samples the same day for better results (within 4-6 hours best). For your other queries let me try to answer them:

1. when we gives the cells to the centrifugation, is it ok with the speed of 1500rpm? is it so strong that makes the cells rupture?

Ans: 1500 rpm is very much fine in fact people do go up to 4000 rpm. Although the timing is important as well. You may centrifuge your cells at 2000 rpm for not more than 5-6 minutes. It is not going to rupture the cells. But after fixation it is advisable to not do it at speeds lower than 2000 rpm.

2. after centrifugation, we dissolve the cells with PBS, should we pipette the cells strongly to make the cells separated?

Ans: You should not do it strongly. you have to be gentle enough to mix the cells and make sure it doesn't cause frothing. The best way is you slowly dissolve the pellet by not aspirating out the entire solution with the pipette and then not pushing it out entirely back to the microfuge tube, and the entire time the tip should be inside the solution. This would result in zero frothing. It sounds naive, but has worked brilliantly so far for me.

3. When we fix the cells, should we add the cell solution into ethanol or we add ethanol into the cell solution? Does this influence the results?

Ans: People have done it either way. I have found better results with the later. I add absolute ice cold methanol, though. While fixing you should ensure that at this step the cell pellet has dissolved homogeneously in minimal amount of residual PBS from the previous decanting step. Moreover, the cells should be mixed thoroughly then and then with the fixative, as keeping it in the fixative for longer time without exposing all the cells with it would result in embarrassing clumps which would seriously affect the acquisition. Doing it like this way would result in better fixation. Fixing with absolute methanol requires a rehydration step with PBS (500 ul @ 4 degrees) after an incubation of a few minutes (3-5 min only) at -20 degrees.

I believe in case you fix it with 70% ethanol you have to incubate it for around 30 minutes, but make sure the fixative is ice cold.

The scale of PI-A needs to adjust to 200 for G1 population cells. In generally, G1 peak is in 200 and G2/M population cells should be around 400 and S phase cells should be in the middle between G1 peak and G2/M peak.

Thanks to you all. Another question, how to optimize the method of sample preparations so the peak of G0/G1 will be sharp and beautiful?

Don't overgrow cells. It is better to harvest cells within 72 hours if you seed cells in a 6-well plate. Remember to use 600ul PBS to suspend the cells first and add 1400 ul 100% cold ethanol drop by drop to fix your cells. You can keep the cells on ice for one hour or a couple of weeks at 4C.

If you want sharper peaks, one problem that occurs is the speed of acquisition. When you run the samples on lower speed you usually get more narrow peaks

I agree with Dr. Ji. Ovrgrowing cells would only lead to cells undergoing stressed conditions with even control cells showing significantly high sub-G1 peak. The no. of cells that need to be seeded depend and therefore defer from cell line to cell line, which you may need to standardize in your lab. Faster growing cell lines require less cells to be seeded.

Moreover, as Dr. Chakravarthy mentioned, the drop wise addition of absolute ethanol over a slow moving vortex, is also a very successful method of fixing cells. Nonetheless it is always very very important to achieve a single cell suspension before addition at each and every step, especially fixing. Sharper and presentable peaks are then destined to follow!

Thanks so much for all the advices, I will try it again with the methods you mentioned, and then give the feedback to you. Thanks a lot and good luck for your research.

Hello everyone, Thanks so much to all of you, with the advices, I get much better results!