PMID- 24055436 OWN - NLM STAT- MEDLINE DCOM- 20141125 LR - 20231111 IS - 1095-9130 (Electronic) IS - 1046-2023 (Print) IS - 1046-2023 (Linking) VI - 66 IP - 2 DP - 2014 Mar 15 TI - Real-time imaging of exocytotic mucin release and swelling in Calu-3 cells using acridine orange. PG - 312-24 LID - S1046-2023(13)00364-2 [pii] LID - 10.1016/j.ymeth.2013.09.004 [doi] AB - Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200 muM) AO solutions presented absorption maximum at 494 nm, emission maximum at 525 nm and only approximately 1.76 ns fluorescence lifetime. By contrast at high concentrations (4-30 mM) favoring formation of AO aggregates, a very different absorption with maximum at approximately 440 nm, dramatically red-shifted emission with maximum at 630 nm, and over 10-fold increased fluorescence lifetime ( approximately 20 ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98+/-0.07x10(-7)c m(2)/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was approximately 10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications. CI - Copyright (c) 2013 Elsevier Inc. All rights reserved. FAU - Shumilov, Dmytro AU - Shumilov D AD - Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, USA. FAU - Popov, Alexander AU - Popov A AD - Research Centre, Centre hospitalier de l'Universite de Montreal (CRCHUM) - Hotel-Dieu, and Department of Medicine, Universite de Montreal, Montreal, Quebec, Canada. FAU - Fudala, Rafal AU - Fudala R AD - Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX, USA. FAU - Akopova, Irina AU - Akopova I AD - Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX, USA. FAU - Gryczynski, Ignacy AU - Gryczynski I AD - Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX, USA; Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX, USA. FAU - Borejdo, Julian AU - Borejdo J AD - Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX, USA. FAU - Gryczynski, Zygmunt AU - Gryczynski Z AD - Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, USA; Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX, USA. Electronic address: z.gryczynski@tcu.edu. FAU - Grygorczyk, Ryszard AU - Grygorczyk R AD - Research Centre, Centre hospitalier de l'Universite de Montreal (CRCHUM) - Hotel-Dieu, and Department of Medicine, Universite de Montreal, Montreal, Quebec, Canada. Electronic address: ryszard.grygorczyk@umontreal.ca. LA - eng GR - 5R21CA149897-02/CA/NCI NIH HHS/United States GR - R21 CA149897/CA/NCI NIH HHS/United States GR - R01 EB012003/EB/NIBIB NIH HHS/United States GR - R01EB12003/EB/NIBIB NIH HHS/United States GR - HL090786/HL/NHLBI NIH HHS/United States GR - R01 HL090786/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20130918 PL - United States TA - Methods JT - Methods (San Diego, Calif.) JID - 9426302 RN - 0 (Fluorescent Dyes) RN - 0 (Mucins) RN - F30N4O6XVV (Acridine Orange) SB - IM MH - Acridine Orange/*chemistry MH - Animals MH - Cell Line, Tumor MH - *Exocytosis MH - Fluorescent Dyes/*chemistry MH - Humans MH - Kinetics MH - Microscopy, Fluorescence MH - Mucins/chemistry/*metabolism MH - Optical Imaging MH - Sus scrofa PMC - PMC4780354 MID - NIHMS526018 OTO - NOTNLM OT - Acridine orange OT - Cystic fibrosis OT - Fluorescence lifetime OT - Mucin granules OT - Spectral shift OT - Total internal reflection fluorescence EDAT- 2013/09/24 06:00 MHDA- 2014/12/15 06:00 PMCR- 2016/03/07 CRDT- 2013/09/24 06:00 PHST- 2013/08/22 00:00 [received] PHST- 2013/09/05 00:00 [accepted] PHST- 2013/09/24 06:00 [entrez] PHST- 2013/09/24 06:00 [pubmed] PHST- 2014/12/15 06:00 [medline] PHST- 2016/03/07 00:00 [pmc-release] AID - S1046-2023(13)00364-2 [pii] AID - 10.1016/j.ymeth.2013.09.004 [doi] PST - ppublish SO - Methods. 2014 Mar 15;66(2):312-24. doi: 10.1016/j.ymeth.2013.09.004. Epub 2013 Sep 18.