Aequorin as Intracellular Ca<sup>2+</sup> Indicator Incorporated in Follicular Lymphoma Cells by Hypoosmotic Shock Treatment

Klabusay, M.; Skopalík, J.; Erceg, S.; Hrdlička, Aleš

doi:10.14712/fb2015061040134

Folia Biologica > Archive > 2015, Vol. 61 > Issue 4 > Aequorin as Intracellular…

Fol. Biol. 2015, 61, 134-139

https://doi.org/10.14712/fb2015061040134

Aequorin as Intracellular Ca²⁺ Indicator Incorporated in Follicular Lymphoma Cells by Hypoosmotic Shock Treatment

M. Klabusay^1,2, J. Skopalík³, S. Erceg⁴, Aleš Hrdlička⁵

¹Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Brno, Czech Republic
²Department of Haemato-Oncology and Department of Internal Medicine – Cardiology, Faculty of Medicine, Palacký University, Olomouc, Czech Republic
³International Clinical Research Center – Center of Biomedical Engineering, St. Anne’s University Hospital, Brno, Czech Republic
⁴Stem Cells Therapies in Neurodegenerative Diseases Lab, Research Centre “Principe Felipe”, Valencia, Spain
⁵International Clinical Research Center – Integrated Center of Cellular Therapy and Regenerative Medicine, St. Anne’s University Hospital, Brno, Czech Republic

Received November 2014
Accepted June 2015

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.

Keywords

intracellular Ca2+ indicator, aequorin, follicular lymphoma, hypoosmotic shock treatment, cell viability.

Funding

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.