Hi all,
I'm doing apoptosis analysis using Annexin V-APC/7-AAD on a Beckman Cytoflex flow cytometer. The band pass for APC is 660/20, and for 7-AAD it is 690/50. Laser delay for 488nm laser is 0, and -36ms for 638nm laser.
I've correctly set compensation using single-stained tubes, and made a quadrant gate according to my negative control sample.
However, cells in my experimental tubes cannot be separated, they are all meshed together. Aren't they should be separated very well given APC and 7-AAD are excited by different laser lines?
This also happens to others using same flow cytometer but with different cell types and drugs. I'm thinking maybe there's something wrong with the machine itself?
Any suggestions?
Thanks in advance.
There seem to be two populations in the lower right graph. If you place your quadrants closer to negative population as I show you in the picture the limit separates 2 populations. Its not a very clear separation but there is some. You can try to graph a scatter plot with less events to see clearer limits.
Hope it helps!
Hey,
We use CytoFLEX for our routine Flow cytometry experiments. We have faced similar problems between 7-AAD (PC5.5 channel - 690/50) and the APC channel (660/20).
7-AAD is excited by the blue laser and has an emission peak (647nm) that is similar to the emission peak of APC (660nm). Thus, the spillover values are significant, making it difficult to visualize distinct populations.
Both APC and 7-AAD are bright dyes having high quantum yield with overlapping emission spectra, resulting in a bad spill, even if the laser delays are increased. Thus, making it hard to compensate. The extent of the spill can be visualized by acquiring single stained samples (7-AAD and Annexin V) in an experiment file keeping both the APC and PC5.5 channels open. 7-AAD will show two distinct positive and negative populations in both channels.
Since more channels are available, it would be advisable to have annexin V on a different colour. Alternatively, PI could be used instead of 7-AAD.
Hi Diego Raffo ,
Thank you for trying to help! Appreciated it!
You were right about gating less events. I ran down population in FlowJo and it shows a little bit better separation between populations.
Hi Pragun Muthya
Your answer is exactly what I need! I suspect the cytometer itself is the cause of the problem and your answer backed me up!
I do get good separations using Annexin V-FITC/7-AAD on same cell line and same treatment.
However, I'm still wondering how others can get well-separated populations using APC/7-AAD on other cytometer models such as BD FACSCalibur or Aria III. I'm guessing it has something to do with the cytometer laser set up.
Thank you for your answer. It was very helpful!
Hi all. I have a question to everyone involved in this conversation. Why compensation is not an issue in single stained tubes containing untreated cells and then become an issue in treated samples?
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