Hi Doorsa,In the past,I had also experienced similar histograms for some of my samples. But i think at least control sample should not be like your histogram. You have not mentioned your cell name.I observed similar (even shift towards subG1) histogram when cells were delicate (MDAMB231 triple-negative breast cancer)or treatment with high concentration of drugs or for longer duration.I used trypsin and 70% chilled ethanol (-20 deg)(Your protocol suggest accutase and 66% ethanol).You can see my protocol at-

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Few suggestions-

1.prior to fixation with -20 deg ethanol, collect cells by centrifugation at as low speed possible.2.Resuspend cells with delicate pipetting 3.Fix with 70%, -20 deg. ethanol 4.DONOT keep at 4 /-20 degree after fixation for more days if the cells are delicate(when i pocessed the sample on the same day instead of keeping for 4-5 days to week(s) in refrigerator,i got better results) 5.Avoid keeping for long hours after PI labelling(2-3 hours at 4deg. is OK ,however PI fluorescence in HeLa cells was intact even when kept at 4 deg.for 12 hrs)

Since some times you got good results with your own protocol, try to repeat exactly the same way.

Hi Doorsa,

I think one of the main issues that both you and Abhinav are having is the presence of RNA in your samples. Propidium iodide will bind to all nucleic acids, and can give histograms like you're seeing. It's common to add 0.2 mg/mL DNAse-free RNAse A to your PI staining solution and then incubate for about 15 minutes at 37°C, 30 minutes at room temperature, or even up to a day or two at 4°C. This should take care of the noise in your histograms.

In my experience with a number of different established cell lines, I could keep cells at 4°C after ethanol fixation for several weeks before staining for flow cytometry and saw no differences in the quality of my results.

Here's a fairly standard protocol for PI cell cycle analysis that you can use as a reference: https://medschool.ucsd.edu/research/moores/shared-resources/flow-cytometry/protocols/Pages/cell-cycle-with-pi.aspx

I haven't tried adding Triton to the PI solution before, but I believe it could be added to ensure that the cells are completely permeabilized for optimal effect of the RNAse.

Good luck!

Hi Doorsa Tarazi

Greetings!!

What type of cells are you working on ? Looks like cells are getting arrested at a particular phase. Or it might be an issue of gating. Probably more carefully the gating should be done to include the cells and exclude the debris. Or, It may also be an issue of RNA as suggested by Nicholas Valerie . PI (propidium iodide) binds with both DNA and RNA, and hence RNase is very much essential to degrade the RNA, so that you get proper histogram output.

Best Regards,

Samrat

Hi everyone,

Thanks for your help. These are patient derived glioma cancer cells but in my experience with them they are not very delicate. I used to use RNAse but stopped at one point because it seemed to make no difference in the quality of data. I will go back to using it and hopefully that will make a big difference. Unfortunately since I don't run the flow myself, I will have to pass the message of more precise gating on to the person who does.

Thanks for all your help!

Doorsa

Dear Doorsa Tarazi ,

You have to always stick to PI-RNAse. As RNAse is very crucial in cell cycle analysis.

All the Best!!

Best Regards,

Samrat