Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells |
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| Institution: | 1. The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin, China;2. Faculty of Mathematics and Physics, University of Ljubljana, and J. Stefan Institute, Ljubljana, Slovenia;1. Department of Ceramics and Glass Engineering and CICECO, University of Aveiro, 3810-193 Aveiro, Portugal;2. Departamento de Fisica, I3N, Universidade de Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal;3. Department of Mechanical Engineering and Centre for Mechanical Technology & Automation, University of Aveiro, 3810-193 Aveiro, Portugal;4. Ferroelectric Research Laboratory, Department of Physics, A N College, Patna 800013, India;1. Department of Chemistry, University of Missouri-Kansas City, Kansas City, MO 64110, USA;2. Department of Chemistry, Kent State University, Kent, OH 44242, USA;1. Centre for Kidney Disease – Venomics Research, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Woolloongabba, Brisbane, QLD 4102, Australia;2. School of Biomedical Sciences, University of Queensland, St Lucia, Brisbane, QLD 4072, Australia;3. University of Queensland Centre for Clinical Research, The University of Queensland, Herston, Brisbane, QLD 4029, Australia;4. Queensland Institute of Medical Research, Herston, Brisbane, QLD 4006, Australia |
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| Abstract: | Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium (Ca2+]c) and nuclear calcium (Ca2+]n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to Ca2+]n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to Ca2+]n and Ca2+]c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells. |
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| Keywords: | Calcium imaging Fluo-3 AM Temperature Brighter nucleus Adherent cells |
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