I have a recurrent issue when analizing apoptosis by flow cytometry using Annexin-Propidium Iodide staining. I can fairly compensate each dye, but when I put the double stained positive control sample, the IP+ polulation "shifts" to the right (see attached images, all of them correspond to compensated samples). I would expect that polulation to ramain in the same place, and that a new double positive polulation appeared in the double positive quadrant. Does anyone know what could be the problem?
My cytometer is a FACS Calibur with an 488nm Argon laser. For this analysis I used FSC vs SSC and FL1 (530/30) vs FL2 (585/42) band pass filters
Any insight will be much appreciated.
The reason why the IP+ population "shifts" in AV-IP dot plots is because of the way that the two dyes, Annexin V and Propidium Iodide, interact with each other. Annexin V binds to phosphatidylserine, which is a marker of apoptosis. Propidium Iodide is a DNA intercalating dye that binds to DNA. When both dyes are present in a cell, they can compete for binding sites. This can cause the IP+ population to shift to the right in the dot plot, as the cells with more Annexin V binding will appear more positive for Annexin V.
There are a few things that you can do to try to minimize this shift:
- Use a lower concentration of Propidium Iodide. This will reduce the amount of competition between the two dyes.
- Use a different laser wavelength. The 488nm Argon laser that you are currently using is not ideal for detecting Annexin V. A 633nm laser is a better option.
- Use a different set of bandpass filters. The 530/30 and 585/42 bandpass filters that you are currently using are not optimal for detecting Annexin V. A 570/20 and 610/20 bandpass filter set is a better option.
All the best
Thanks for your reply. What do you mean bye competition for binding sites? They actually bind to different molecules and regions within a cell.
Regarding the optical configuration, the cytometer has only one (488nm) laser. In this case, Annexin is bound to FITC (ex max 495nm), as shown in the spectrum image I attached.
Dear Pablo,
By the first plot, I can infer that the median fluorescences are not ajusted to zero. Dos you check the medians before and after compensation? It is possible that your cells haver a particular autofluorescence patterns that may be interfering in the compensation. Also, there are other channel (FL) for IP?
Felipe Andrés Cordero da Luz Hello. Maybe I should've posted an uncompensated plot too so it would've been more clear. What do you mean by "Fluorescence adjusted to zero"?
Dear Pablo,
I am sorry. The correct is to set the Median Fluorescences Intesities (MFIs) of the spillover channel similar to the non-labaled one. For example: ther is a spillover of FITC (FL1) in the IP Channel (FL2), and a correct compensation would set the MFI of FL2 of the AV (single-labeled) similar of a non-labeled one. I am attaching a document that explanou a lot of this issue. Hope this can help.
Felipe Andrés Cordero da Luz yes, sure. I usually do compensations. The problem is with this particular case. The IP+ polulation seems to shift to a AV+ region. It seems as if the emission of FITC (AV) would alter the emission spectrum of IP, so that it spills more into FL1 channel, or something like that
Pablo,
I am sorry, bit in this case I do not know another possible cause rather thank undercompensation.
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