Dear Karim, I really like your question. First because I see a lot of people stating that in cases of autofluorescence we should adjust the voltage for fluorescence on the cytometer in order to bring the negative (isotype control) to the first decade. In another words, people tend to perform instrument settings using their own cells regardless the autofluorescence component. I disagree with that since I always recommend my students to set the instrument using a calibration system (like CaliBrite form BD, or other available). As far as your question, I would take the first alternative, based on the results you present. However, it can also be result of a low expression of the marker X and in this case to take the 9īs question (to confirm the hypothesis) I suggest you to use a more sensitive fluorochrome, such as PE or Cy-5, in a biotin-Streptavidin system to increase the sensibility of your test. I have experience working with eosinophils, neutrophils, monocytes from in vitro culture and PFA-fixed T. cruzi epimastigotes, which show high autofluorescence. However, I always find then to shift to a higher MFC (mean fluorescence channel) when they express any X marker I am looking for. I hope it helps. Thank you for sharing your experience. Best regards, Olindo Assis
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