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Three peaks in cell cycle using PI

Three peaks in cell cycle using PI - Flow Cytometry Forum

Three peaks in cell cycle using PI - Flow Cytometry Forum. Discuss FACS cell sorting, flow cytometry, analysis and cell staining.


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Old 03-23-2012, 11:49 PM
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Default Three peaks in cell cycle using PI



Hi all,

we have three peaks when doing cell cycle analysis using propidium iodide.

We are looking at U87 glioblastoma cell lines
(ATCC: HTB-14), for which the ATCC info sheet states: "This is a hypodiploid human cell line with the modal chromosome number of 44 occurring in 48% of cells. The rate of higher ploidy was 5.9%."

The mean channel number of the second peak is about 2x of the first one and the third 2x of the second. So it looks like cells with 2n / 4n chromosome number, right? How can we then look for apoptosis by DNA fragmentation (sub-G1)?

Any help is greatly appreciated!

Karl
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Old 03-23-2012, 11:51 PM
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Default Re: Three peaks in cell cycle using PI

I need 5 posts to be able to post a picture.... Post 2.
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Old 03-23-2012, 11:51 PM
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Default Re: Three peaks in cell cycle using PI

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Old 03-23-2012, 11:52 PM
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Old 03-23-2012, 11:54 PM
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Default Re: Three peaks in cell cycle using PI

The histogram looks like this, we did some FlowJo curve fitting, and you can see the modeling of the 2nd and 3rd peaks. But how to deal with the first one? Sub-G1 events just below the 2nd peak would coincide with the area between the 1st and the 2nd peaks, which would be interpreted as S phase if the 1st peak is G1...
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Old 03-23-2012, 11:57 PM
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Default Re: Three peaks in cell cycle using PI

We also did doublet discrimination, so all doublets should be gated out...
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Old 07-17-2012, 02:50 AM
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Default Re: Three peaks in cell cycle using PI

I have the same problem, so I'm wondering if you've found a explanation or solution since you've encountered this?

Would really appreciate some help on this.
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Old 06-11-2013, 03:30 PM
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Default Re: Three peaks in cell cycle using PI

you could try increasing your threshold.
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