Rational method in the repetitive calcium oscillation measurement in wild type human epithelial kidney cells

Abstract

Cells stimulated with physiological stimuli usually exhibit oscillations in cytosolic Ca(2+) concentration ([Ca(2+)](i)), a signal playing central roles in regulation of various cellular processes. For explicating their unknown mechanisms, studies are commonly conducted in single cells from several cell lines, in particular the human epithelial kidney (HEK293) cell line. However, [Ca(2+)](i) oscillating responses to agonists in vitro are found difficult to be induced and varied with different types of cells and agonists. This study shows that treatment of the wild type HEK293 cells with low concentrations of carbachol (1-10μM), an agonist of the muscarinic receptor, resulted in non-oscillated but sustained [Ca(2+)](i) increase by loading the cells with 1 μM fura2/AM. However, repetitive and long lasting [Ca(2+)](i) oscillations could be induced in 31.1% of the tested cells loaded with 0.1μM fura2/AM. Additionally, the occurrence of the typical Ca(2+) spikes further increased to 47.2% and 60.7% when the Ca(2+) concentration in the bathing medium was decreased from 1.8 mM to 1.5mM and the medium temperature was set to 35±1°C from 22±2°C. Therefore, this study provides a useful approach for measuring [Ca(2+)](i) oscillatory response to relevant physiological stimulation in a wild type cell line through the adjustments of the concentrations adopted for the Ca(2+) indicator and extracellular medium Ca(2+) and of the temperature set for the experiment.