I used OneComp eBeads (eBioscience) to set up the compensation for PE & PE-Cy7. However, in the single PE staining tube, I couldn't achieve two distinct populations with similar mean values. I tried to increase the voltage for PE and I just got one population, but when I reduced the voltage, then the negative population shifted too much to the left side.
Additionally, I still got the spill-over between PE & PE-Cy7. After having increased the spectral overlap, I got the figures like the last one but I thought the signals for PE were too dim. Am I doing it right? Could you please give me any advice to obtain an optimal compensation?
Thank you for any help you may provide
In all panel we can see negative events below 102. I think your negative events are here. You should use the biexponential transformation and you will see your negative events.
Best,
Matthieu
In all panel we can see negative events below 102. I think your negative events are here. You should use the biexponential transformation and you will see your negative events.
Best,
Matthieu
You can increase your voltage a bit but I am not sure it is necessary. Take care that your positive events do not exceed 105. In addition your compensation seem not too bad
Hello
i agree with Matthieu Rousseau .Also , find links here and hope help you.
Good Luck
https://www.med.unc.edu/flowcytometry/resources-2/compensation
https://www.ebioscience.com/media/pdf/literature/multicolor-flow-cytometry-tech-resource-guide.pdf
http://www.ebioscience.com/resources/faq/flow-cytometry-faq.htm
https://depts.washington.edu/flowlab/Cell%20Analysis%20Facility/BD%20Hotline%20Selecting%20Reagents%20for%20Multicolor%20Flow.pdf
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