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