Till polychrome 5 digital imaging system

OLE cells were grown on 25 mm circular coverslips and incubated with the fluorescent Ca²⁺ indicator Fura-2AM (2 μM) for 1 h at RT in normal extracellular solution (NES) (137 NaCl mM, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM HEPES and pH adjusted to 7.4 by NaOH). Coverslips were washed and incubated with NES for 30 min at RT in the dark. The coverslips were placed in a stainless-steel holder (bath volume ∼0.8 mL) and imaged using a Leica DMI300 B inverted microscope coupled to a TILL Polychrome V digital imaging system (Toptica Photonics, Farmington, NY, USA). Cells were treated with flow-through of 1 µM P4 for 100–400 seconds, 10 nM for 500–700 s, and 80 mM KCl at 800 s. In parallel, a second coverslip of cells was pretreated with 10 nM nicardipine for 30 min followed by the same treatment paradigm described previously. Results present the ratio (R/R₀) of fluorescence intensities at the excitation wavelengths of 340 and 380 nm. Ca²⁺ imaging data were analyzed using Origin 2020 Software (Origin Lab, Northampton, MA, USA), and data for all figures are expressed as mean ± s.e.m. Statistical significance was calculated using one-way ANOVA followed by Student’s t-test and ** represents statistical significance (**P < 0.01).