title: Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells.
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 ([Ca(2+)]c) and nuclear calcium ([Ca(2+)]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 [Ca(2+)]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 [Ca(2+)]n and [Ca(2+)]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.
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin, China.
authors.forename: Guixian
authors.initials: G
authors.lastname: Meng
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin, China. Electronic address: plt@nankai.edu.cn.
authors.forename: Leiting
authors.initials: L
authors.lastname: Pan
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin, China.
authors.forename: Cunbo
authors.initials: C
authors.lastname: Li
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin, China.
authors.forename: Fen
authors.initials: F
authors.lastname: Hu
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin, China.
authors.forename: Xuechen
authors.initials: X
authors.lastname: Shi
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin, China.
authors.forename: Imshik
authors.initials: I
authors.lastname: Lee
authors.affiliation: Faculty of Mathematics and Physics, University of Ljubljana, and J. Stefan Institute, Ljubljana, Slovenia.
authors.forename: Irena
authors.initials: I
authors.lastname: Drevenšek-Olenik
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin, China.
authors.forename: Xinzheng
authors.initials: X
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin, China.
authors.forename: Jingjun
authors.initials: J
authors.lastname: Xu
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D000818:Animals; D002448:Cell Adhesion; D002467:Cell Nucleus / Q000378:metabolism*; D004951:Esterification; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008954:Models, Biological; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D013194:Staining and Labeling; D013696:Temperature; D013997:Time Factors; D014966:Xanthenes / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D014966:Xanthenes; C059715:Fluo-3
keywords: Adherent cells; Brighter nucleus; Calcium imaging; Fluo-3 AM; Temperature