Rc 42lp

The filtered cells were incubated for 20 min at 37 °C with 10 μm Rhod‐3, AM (R10145, ThermoFisher Scientific) and 0.04% Pluronic‐F127 (P2433; Sigma‐Aldrich) contained aCSF in a bath chamber (RC‐42LP; Warner Instruments, Hamden, CT, USA). After Rhod‐3, AM incubation, chamber was placed on a stage of inverted microscope (NIKON ECLIPSE Ti, Tokyo, Japan) then washing with aCSF (room temperature) for 5 min before lyral application. The aCSF and lyral were perfused through tubing with a peristaltic pump at a flow rate of 2 mL·min⁻¹. 100 μm lyral diluted with 0.1% DMSO in oxygenated aCSF was applied for 15 s. After treatment of lyral, mixture solution of 100 μm IBMX and 10 μm forskolin was applied for confirmation of neuronal viability. The intracellular Ca²⁺ level was measured by 610 nm, emission of Rhod‐3, AM fluorescence ratio (excitation, 545 nm). Emitted fluorescence was recorded every 2 s using CCD camera (Andor Technology, Belfast, UK). Analysis for intensity of fluorescence was carried out using MetaFluor (Molecular Devices, Sunnyvale, CA, USA). All pseudocolor images were converted from fractional fluorescence change (ΔF/F, Δ[Ca²⁺]_i), corresponding to changes in intracellular calcium ion concentration.

Fluorescence measurements were obtained using IX71 inverted microscope (Olympus, Tokyo, Japan) attached with a 60× (numerical aperture 0.9) water-immersion objective lens as previously reported^{14 (link)}. The signal was detected by a cool CCD video camera Ixon (Andor Co., Tokyo, Japan) coupled to Lambda-10.2 (Sutter Instruments, CA), and excitation was provided by Sutter DG-4 and 175-W Xenon-Arc lamp (Sutter Instruments, Navato, CA). For the experiments, cover slips were placed in an imaging chamber (RC-42LP and RC-43C; Warner Instruments, Hamden, CT) mounted on the stage of the inverted microscope and maintained at 37 °C with TC-344B thermo-warmer for chamber and TA-29 thermo-warmer for perfusate (Warner Instruments, Hamden, CT). The changes in fluorescence intensity were quantified using MetaFluor imaging software (Universal Imaging Co., Ypsilanti, MI) for each experiment. To exclude the possibility of selection bias of the measured MCs, the fluorescence intensity of all living mesangial cells in each cover glass was measured.