title: Aequorin as Intracellular Ca2+ Indicator Incorporated in Follicular Lymphoma Cells by Hypoosmotic Shock Treatment.
abstract: Natural proteins can be used in measuring intracellular Ca(2+) concentration. As one of the Ca(2+)- regulated photoproteins, aequorin has several advantages in comparison to widely used Ca(2+) fluorescence indicators (e.g., fura-2, indo-1 and fluo-3), including high dynamic range and resistance to motion artefacts. However, incorporation of aequorin into cells remains a challenge. Hypoosmotic shock treatment was optimized and used as a method for loading aequorin into the cytoplasm of follicular lymphoma cells. Measurement of aequorin luminescence in the cells was performed using a luminometer with a sensitive photomultiplier tube and the luminescence intensity was recalculated into intracellular [Ca(2+)]. The value of (0.85 ± 0.52)·10-6 M was found. We show that the optimized method of incorporation was effective for loading aequorin into follicular lymphoma cells in vitro. The cell viability remains high immediately after the procedure. This method can also be used for measuring intracellular Ca(2+) concentration in other types of non-adherent cells.
authors.affiliation: Department of Haemato-Oncology and Department of Internal Medicine - Cardiology, Faculty of Medicine, Palacký University, Olomouc, Czech Republic.
authors.forename: M
authors.initials: M
authors.lastname: Klabusay
authors.affiliation: International Clinical Research Center - Center of Biomedical Engineering, St. Anne's University Hospital, Brno, Czech Republic.
authors.forename: J
authors.initials: J
authors.lastname: Skopalík
authors.affiliation: Stem Cells Therapies in Neurodegenerative Diseases Lab, Research Centre "Principe Felipe", Valencia, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Erceg
authors.affiliation: International Clinical Research Center - Integrated Center of Cellular Therapy and Regenerative Medicine, St. Anne's University Hospital, Brno, Czech Republic.
authors.forename: A
authors.initials: A
authors.lastname: Hrdlička
mesh_terms: D000331:Aequorin / Q000378:metabolism*; D000814:Aniline Compounds / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D006801:Humans; D007211:Indoles / Q000378:metabolism; D008224:Lymphoma, Follicular / Q000378:metabolism*; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D007211:Indoles; D014966:Xanthenes; C059715:Fluo-3; D000331:Aequorin; C048960:indo-1; D002118:Calcium