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. 2022 Mar 15;10:827987. doi: 10.3389/fbioe.2022.827987

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Copyright © 2022 Wanner, Barnhart, Apostolakis, Zlojutro and Asosingh.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Improved resolution of fully stained sample with AF included in unmixing. AF was either included or not included in the unmixing of the fully stained sample. By including the AF in the unmixing, the resolution for BUV395 was enhanced, enabling for better detection of the non-hematopoietic/non-EC fraction (CD45-/Sca-1-; yellow gate) (A). Increased resolution for AF532 was also seen, allowing for the detection of eosinophils (B). The absence of AF in the unmixing masked the signals of PE and BV605 which reduced the resolution between these fluorophores and lead to false positives of epithelial goblet cells (C). The poor identification of the non-hematopoietic/non-EC subset in (A) resulted in the loss of events downstream in the gating strategy as seen by a reduced α-SMA + population (D).