Ix81 inverted light microscope

Cells were seeded into a Corning 8.0 uM pore transwell chamber (Sigma-Aldrich) at 5 ×10⁴ with 200uL of Dulbecco’s Eagle Minimum Essential Media. A total of 500uL of media was placed inside the bottom well. Cells were incubated at 37°C with 5% CO₂ for 48 hours and then fixed with formaldehyde. Once non-migrated cells were removed from inside the chamber, fixed cells were stained with crystal violet and membranes imaged using an Olympus IX81 Inverted Light Microscope. All images were taken at 10X magnification. 12 non-overlapping images from each membrane were captured and used with ImageJ64 (version 10.2) to measure the percentage area of migrated cells. Experiments were carried out in biological triplicate, and any outliers removed prior to statistical analyses.

Rat-1 cells expressing the ΔN or ΔNΔC α_1D-adrenoceptors were transfected with a cytoplasmic extracellular signal-regulated kinase activity reporter plasmid (EKAR, EGFP-mRFP) (Harvey et al., 2008 (link)) using Lipofectamine 2000. EKAR is a genetically encoded, fluorescence resonance energy transfer-based sensor of ERK activity. This reporter contains an EKR substrate peptide, an ERK docking domain, a phospho-binding domain, and, at the amino and carboxyl termini, mRFP and EGFP, respectively (Harvey et al., 2008 (link)). ERK substrate peptide phosphorylation, by the endogenous protein kinase, triggers a reporter conformational change, which allows proximity of the fluorescent proteins and fluorescence resonance energy transfer (FRET) (Harvey et al., 2008 (link)). Confocal images were obtained 48 h post transfection using an Olympus Fluoview FV 100 laser confocal system attached/interfaced to an Olympus IX81 inverted light microscope with a ×60 water immersion objective; EGFP was excited at 488 nm and emission was simultaneously recorded at the green (500–530 nm) and red (555–655 nm) channels. Confocal images were viewed and processed employing FV10-ASW 1.6 software (Olympus).

Cells cultured on glass-bottomed tissue culture dishes were washed once with Normal Tyrode’s buffer and incubated with 2.5 μM Fura-2AM (Invitrogen) in loading buffer with 2% BSA for 20 min at room temperature. Cells were then washed twice with Normal Tyrode’s buffer and incubated for another 10 min at 37°C to allow for de-esterification of Fura-2AM. To record the changes in cytosolic Ca²⁺ transients, images were acquired continuously, from 3 min before and at least 10 min after PDGF treatment using an Olympus IX81 Inverted Light microscope. The cells were excited alternatively at 340 and 380 nm. Fluorescence signal intensity was acquired at 510 nm. Data are presented as AUC and peak amplitude. To estimate baseline cytoplasmic Ca²⁺ levels, two adjacent chambers were created on a glass bottom microwell dish by applying a divider of vacuum grease. Then, VSMCs transfected with MCU siRNA or scrambled control were seeded in the two chambers without mixing the two cell preparations. Fura 2-AM imaging was performed as described above.