I am using Annexin V-FITC to analyze apoptosis by flow cytometry. Currently, I am working with the two neuroblastoma cell lines SK-N-BE(2) and SK-N-AS.
The assay works perfect using the neuroblastoma cell line SK-N-AS. Only a small number of control cells are Annexin V positive. When I add doxorubicin, the number of Annexin V positive cells increases.
However, a huge number of SK-N-BE(2) control cells (untreated cells) are Annexin V positive (more than 60%) in spite of the fact that cells are looking well. Has someone observed similar effects ? Is SK-N-BE(2) probably not suitable for this assay?
Hi
I have found that Annexin V staining can result in negative cells being pulled to the right, so that they appear to be expressing low levels of Annexin when in fact they are not truly positive. especially with cell lines and cells in extended culture. I have found that when PI is added this resolves the positon for applying quadrants to resolve the us, annexin+PI-, Annexin+PI+ and Annexin-PI+ populations.
To test whether this is the case for your cell line I would run 4 samples:
unstained control
cells stained with annexin only
cells stained with PI only
cells stained with Annexin and PI
analyse them all on Annexin v PI plots and look at where the PI cells appear in the plots when analysing cells stained only with PI, and use this to set the quadrant for looking at annexin staining.
If you also do this with the line which works it should give the same result, that using the PI to set the quadrant does define the cut off between Annexin+ and Annexin-.
Y ou could also use a different method of assessing apoptosis eg hypodiploid peak to convince yourself that the PI gating is correct for determining the annexin status of cells.
cheers
kay
Hei,
thank you very much for your answer! Unfortunately, I cannot use PI as I also treat cell with doxorubicin.
Doxorubicin exhibits intrinsic fluorescence (excitation wavelength, 480 nm; emission wavelength, 545-650 nm). Thus, the spectral range of doxorubicin significantly overlaps with that of PI. In concordance to this, I found that PI cannot be used when doxorubicin-treated cells are analyzed for apoptosis by flow cytometry. Here, I found that doxorubicin-treated cells exhibit significant higher “autofluorescence” as compared to untreated cells.
Which other methods may be easy to establish and to perform?
Possibly you can use HOEST or DAPI for control. I think it can help.
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