It's quite surprising to see a difference. However, how do you confirm whether cells are in G0 or G1 phase? You can also check for the cell size. More cell size produces more autofluorescence. You may also try other dyes such as Propidium iodide etc. Check for a standard protocol for propidium iodide below. Good luck

http://www.abcam.com/protocols/flow-cytometric-analysis-of-cell-cycle-with-propidium-iodide-dna-staining

Thank you for your reply! I meant that G0/G1 or S or G2/M fluorescence should be at the same levels for all my samples and not shifting along the 7-AAD axis. So what I see it is a shift of my histogram along my samples.

I got your point now. I assume the dye binding is not uniform among your samples. You need to make sure that the sample volume and the number of cells in the sample are equal in all samples. You can also try using different concentrations of the dye. In your case, I guess you need to go up. I hope this information helpful to you. Goodluck

Thank you! I got a good starting point to work on it!

HI Mara Esposito,

We generally use Propidium Iodide for Cell cycle analysis. If you are using suspension cells you need to wash them once with PBS and then add Propidium iodide with triton X 100, if you are using adherent cells you need to detach them with accutase instead of trypsin. For flow cytometry gating, if you are using BD Calibur,  first select cell population based on size and granularity, then do doublet discrimination with FL-3A VS FL-3W, then a histogram on FL-2 height. 

Increased cell size represent that your cell may be dying by necrosis.

Hope this answer will help you,

Best Luck

Gajendra

Dear Mara Esposito ,

could you give us the protocol you are using? It is very unusual to not have a good G1/G0 vs G2 discrimination, regardless of your cellular system. Kind regards, Harrie Verhoeven.

Dear Mara, is the sample fluorescence variability completely random between the samples? Sometimes during experiments with consistent number of samples it is possible to detect a significant and linear fluorescence decrease from the first acquired sample to the last. This could be attributed to the bleaching dye. If you do not protect the tubes from light during the acquisition and you have to acquire several samples, taking you more than 15' - 20' the bleaching could significantly affects your experiment in the way you described (a whole histogram shift). Obviously, more logs of fluorescence you lost, more difficult will be the population discrimination. If it could be the case, I suggest wrapping the tubes with tinfoil and keep samples in the dark until the acquisition. Hope this could help, good luck

Simone

Dear Harrie,

the protocol the I am using is the following:

Reagents

FACS buffer (1x PBS; 1% BSA; 0.09% Sodium Azide)

FACS staining solution (1x FACS buffer; 5% 7-AAD)

Protocol

1.       Harvest the cells with trypsin

2.       Wash with 5 ml cold PBS 1X

3.       Resuspend in 500 µl 1x PBS (pipette up and down)

4.       Fix cells with 4.5 ml ice cold 70% ethanol. Add drop wise to the pellet while vortexing. This should ensure fixation of all cells and minimize clumping.

5.       Incubate for 30’ at 4ºC or store indefinitely at 4ºC.

6.       Wash x2 in PBS (1ml). Spin down at 850g for 5’ and be careful to avoid cell loss when discarding the supernatant especially after spinning out of ethanol.

7.       Count the cellsà 106 cells for each sample

8.       Resuspend the cells in 100 µl FACS staining solution (per sample: 95 µl buffer + 5 µl 7-AAD à 0.25 µg 7-AAD)

9.       Incubate for 15 min at RT.

Do you see any mistakes in my procedure?

Thank you for your feedback!

I send also my results with the histograms where you can see the shift along 7-AAD axis.

Please, let me know what you think!

Hi Mara Esposito,

By seeing your experimental result I feel you have technical problem in your experiment. Check for the density of cells used during experiment. If cell density is higher and it is grown you will see histogram for 24hrs of your experiment. Shift in peak may be associated with tube to tube variation of 7AAD. the tube to tube variation may be due to incomplete removal of supernatant before addition of 7AAD.

I would also like to suggest that during acquisition of DNA Data you place your G1 peak at around 100k, this will help you for looking at Sub G1 peak a marker of Apoptosis ( Fragmented DNA) As seen in your experiment (U2OS cells synchronized in G2/M by STLC for 16h). Therefore in this experiment you don't need unstained control for setting voltage. you can set voltage with experimental control.  

Hope this will help you,

Good Luck

Thank you for your reply Gajendra. When do you suggest to count the cells? Before ethanol fixation or after?

It is not about counting the cells before or after ethanol fixation, you should count the cell at the time of performing stimulation (0hr time point). you need to have equal number cells in control and treatment. check for  seeding density of your cell line. 

If you can use high staining volume, the cell count does not matter anymore. If you would like to avoid counting, you can just increase the staining volume.

Dear Mara Esposito ,

I have studied your supplied information. Why are you using 7AAD as a DNA stain? And what instrument are you doing the analysis on? From the graphs you provided, I conclude that the acquisition parameters were not identical for the different graphs. The use of a fixation step is often problematic. What I used to do, is release the nuclei in a suitable buffer and stain them with PI. PBr or DAPI. In this way it is possible to obtain very low CVs, in the 1 % range, with plant cells. With animal, human cells, one could even get lower CVs. Your diagrams do not come close, but the variation in the absolute position on the X-axis is really worrying. I will try to help you if you can provide more details. Kind regards, Harrie Verhoeven