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everyone.

Since I am new in the flow cytometry technique, I am not really sure how to analyze the result. We use cell U-937 Lymphoblast.All Cells FSC - SSC

Could Anyone help to explain me this one?

Thanks in advance!

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  • $\begingroup$ Please explain what your study is about. We can't just guess what you are doing $\endgroup$
    – Flo
    May 29, 2017 at 16:48

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Disclaimer: Assumes you know how flow cytometry works.

You've done a flow-based AnnexinV apoptosis assay here.

Annexin A5 (AnnexinV) protein binds to phosphatidylserine (PS), which is typically in abundance of the inner leaflet of live cells. Upon the initiation of apoptosis, PS is known to flip to the outer leaflet. Thus, you can use AnnexinV conjugated to a fluorescent molecule such as fluorescein isothiocyanate (FITC) to look at externalized PS.

7-Aminoactinomycin D or 7-AAD is a fluorescent intercalator that can be used to detect DNA and is excited on the 488nm laser (emits on a 650nm longpass filter or the like). The dye is not permeable to live cells, and so for dead or dying cells with compromised membrane permeability, it binds freely to DNA.

The problem with either alone is you cannot say a 7-AAD+ cell is apoptotic and you cannot say an AnnexinV+ cell is apoptotic, because we know that you have early apoptosis and late apoptosis, and in either the cell isn't wholeheartedly dead, yet. You also have necrosis.enter image description here

And so on a dot plot of 7-AAD and Annexin V, you can create 4 quadrants based on the mechanism of the two components:

Double-negative: No external PS or DNA binding means these are viable cells.
Double-positive: Both external PS and membrane instability means late apoptosis.
AnnexinV single-positive: Undergoing early apoptosis, nucleus isn't yet permeable.
7-AAD single-positive: Completely dead or necrotic, membranes have probably disintegrated.

The basis of the assay is you're trying to determine at what stage of cell death your cells are in.

The unstained control is a way of looking at your cells alone, and allows your to correct for autoflorescence, florescence generated by your cells just from excitation alone. You also want to run single-stain controls to see where your positive, and negative populations lie. Without FMO (fluoresence minus-one, basically your full staining panel minus a color) controls, it's hard during gating to confidently say where your positives and negatives are delineated, due to the effects of spillover and noise.

A note about the AnnexinV single-stain control: You can see that in the untreated sample, there's basically no AnnexinV, but in the control it's 22.0% of the cells. Single-stain controls where you assume positives exist have to have positive and negatives. Without positives, where do you set the gate for them? What if the population shifts during acquisition? They probably used cells that were dying on purpose to generate confident controls for that sample.

That's the left half of your data. The right half are treatments. Basically you've stained the cells for both markers and then treated them with Torin2, Rapamycin and Mitomycin to see how those compounds affect the viability of the cells. There's a control where you added nothing.

Based on the gates set with the controls, you can see that the mitomycin sample has the greatest effect: The cells are 6.54% AnnexinV positive, undergoing early apoptosis, and 14.3% are double-positive, undergoing late apoptosis, and 8.09% are dead or necrotic.

The conclusion you'd derive is that mitomycin, but not the other compounds, has a profound effect on the viability of the cells.

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  • $\begingroup$ Thanks So much for your explanation. I did learn a lot from you. :-) @CMosychuk $\endgroup$
    – Sokviseth
    May 30, 2017 at 8:34
  • $\begingroup$ And in the 7-AAD single staining, we did not put Annexin V, why we can see the amount of cell death in the annexin V quadrant? $\endgroup$
    – Sokviseth
    May 30, 2017 at 14:52
  • $\begingroup$ The laser is still collecting in the annexin channel, the author simply didn't stain that marker. So it could just be noise, or autofluorescence, or spillover in some cases (I don't know what fluorophore they used to stain annexin). If you saw something significant there, you could subtract that background from your treatments to get a more accurate readout. $\endgroup$
    – CKM
    May 30, 2017 at 18:44

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