Author:
Klabusay M.,Skopalík J.,Erceg S.,Hrdlička Aleš
Abstract
Natural proteins can be used in measuring intracellular Ca2+ concentration. As one of the Ca2+- regulated photoproteins, aequorin has several advantages in comparison to widely used Ca2+ 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 [Ca2+]. 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 Ca2+ concentration in other types of non-adherent cells.
Funder
European Regional Development Fund
Ministerstvo Zdravotnictví Ceské Republiky
Publisher
Charles University in Prague, Karolinum Press