Aequorin as Intracellular Ca 2 + Indicator Incorporated in Follicular Lymphoma Cells by Hypoosmotic Shock Treatment ( intracellular Ca 2 + indicator / aequorin / follicular lymphoma / hypoosmotic shock treatment / cell viability )

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. Received November 16, 2014. Accepted June 24, 2015. This work was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic, Grant No. NS 9670-4, and by the European Regional Development Fund, Contract Grant (Project) FNUSA-ICRC, CZ.1. 05/1.1. 00/02.0123. Corresponding author: Aleš Hrdlička, International Clinical Research Center – Integrated Center of Cellular Therapy and Regenerative Medicine, St. Anne’s University Hospital, Pekařská 53, 656 91 Brno, Czech Republic. Phone: (+420) 543 185 524; e-mail: ales.hrdlicka@fnusa.cz Abbreviations: GFP – green fluorescent protein, HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HOST – hypoosmotic shock treatment. Introduction Intracellular [Ca2+] in B lymphocytes has been studied in the past decade with a focus on the details of cellular processes in leukaemia and lymphoma cell signalling (Feske, 2007). Numerous techniques for analysing cellular or subcellular [Ca2+] have been established. Each method has its advantages when monitoring one or more types of cells or cellular processes. Fluorescent probes (fura-2, indo-1, fluo-3, fluo-4) are widely used synthetic indicators of intracellular Ca2+ concentration in B lymphocytes. Calcium-regulated photoproteins have been used to monitor intracellular Ca2+ for more than 30 years (Vysotski et al., 2006). Aequorin, a 21.4 kDa bioluminescent protein that has been studied extensively since its discovery nearly 50 years ago (Shimomura et al., 1962; Shimomura, 1995), has numerous advantages over other indicators, in particular high signal-to-noise ratio, high dynamic range and resistance to motion artefacts. Because of its molecular weight, it is difficult to load aequorin into cells, while on the other hand, its diffusion to other cell compartments is very limited. Aequorin cannot spontaneously penetrate into cells through the lipid membrane as can acetoxymethyl esters of fluo-4 or indo-1. Aequorin can be loaded into cells by the methods of microinjection, diffusion from patchclamp pipette, and lipotransfer delivery or hypoosmotic shock treatment (HOST). Transfection with vectors of aequorin is greatly advantageous, because functional aequorin can be regenerated by subsequent treatment with coelenterazine. Microinjection has been used especially for neuronal cells (Meech, 1981), diffusion from patch clamp for neuronal and muscle cells, and lipotransfer delivery (Hallett and Campbell, 1982; Barber et al., 1996) as well as centrifugation (Borle et al., 1986) for several types of cells. HOST has been used espe-