Hello,

Can you please provide your sample preparation protocol? Most include alcohol-based fixation followed by RNAse treatment to ensure that the propidium iodide only binds to DNA, which allows for cell analysis based on relative DNA content (2n-4n). If possible, it may also be helpful to include a pharmacological control such as hydroxyurea or nocodozol to block cell cycle progression at a specific stage, S and M, respectively. The assay should be sensitive enough to detect these changes in cell cycle frequency.

Hello Muhammad, You do indicate what type of instrument you are using? This application tech note for the FACSArray maybe of use BD_Research_Cell_cycle_FACSArray_AppNote

Andrew Lewis

Martin Waterfall Thank you for the suggested material. I am using SYSMEX cube 6 for flow cytometry.

Alex Abel Thank you for the suggestions. I fix the cells with 70% ethanol and then use RNase treatment as well.

Hello Muhammad,

• I've not use the SYSMEX cube but they claim a fluorescence sensitivity: ≤ 100 MESF (FITC) | ≤ 50 MESF (PE) which is comparable with high equipment(BD Fortessa) fluorescence sensitivity: ≤ 80 MESF (FITC) | ≤ 30 MESF (PE). I would expect a clean signal from PI labelled cells fixed with EtOH, especially if supplemented with RNase. Secret is to make sure you have a single cell suspension before slowly adding the EtOH with gentle mixing. Best of luck

Andrew Lewis

1. i want to ask if when constrained the g1/2 peak, a grey bar like appears, but never goes away even i when copied it MS word. so how can i solve it.

2.is this data relevant? (image)

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this is ctrl plus 3x replicate results (image), is it valid and comparable result to conclude KD affects cell cycle distribution? no matter i tried to understand it, it was not easy [ my first experience to flow cyto data analysis]. my primary objectives mainly focus if there is a cell cycle disruption as gene KD experiment.

thank you in advance.