2.3. Intracellular calcium imaging

Fura-2 loading and experimental protocols performed were similar to those performed as described previously [28]. Briefly, cells were plated and grown in an 8-well chamber, washed twice with 1:1 PBS:FBS solution, and incubated at 37 °C in 5% CO₂ for 30 min in 3 μM Fura-2 in Ringer's solution (NaCl 150 mM, glucose 10 mM, HEPES 5 mM, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, pH 7.4). The cells were washed with fresh Ringer's solution, and cells were allowed to equilibrate for 30 min prior to imaging. For experiments studying plasma membrane Ca²⁺-ATPase (PMCA) inhibition, fresh Ringer's solution was removed and replaced with a Ca²⁺-free buffer, Ca²⁺- and Mg²⁺-free Hank's Balanced Salt Solution (HBSS). The PC-3 cells were imaged with an Olympus IX51 inverted microscope. Treatments were added manually in 100 μL volumes as outlined in the results section of this paper.

Images acquired were analyzed using CellSens software (version number 1.11, Olympus, Tokyo, Japan) from Olympus. At least 10 cells per experiment were chosen and analyzed in order to measure changes in relative [Ca²⁺]_i. The results of the analysis were normalized for each treatment and used to find the mean change in relative [Ca²⁺]_i for each experiment. Each experiment was performed in triplicate (n ≥ 3). The calcium traces were quantified by determining the area under the curve (AUC) or total calcium response after treatment addition using GraphPad Prism 7 (La Jolla, CA, USA).