This article details a method for measuring the contractile response of aortic smooth muscle cells (SMCs) using an electrical cell-substrate impedance sensing (ECIS) system. The protocol outlines the preparation of SMCs, their stimulation, and the subsequent measurement of their contraction in real-time.
The aorta wall predominantly has smooth muscle cells, or SMCs, containing contractile protein machinery that contract and relax to regulate aortic blood flow.
To measure the contractile response of aortic SMCs in vitro, obtain a multi-well plate containing an electrical cell-substrate impedance sensing, or ECIS, sensor array. Each array contains gold film electrodes at the well bottom, delineated with an insulating film. Coat the wells with gelatin.
Seed primary human SMC suspension into the wells. Transfer the ECIS-containing well plate to a plate holder connected to an amplifier and data acquisition system while ensuring electrode contact with the holder. Apply small constant alternating current between the electrodes to measure the potential across them.
During incubation, SMCs adhere and spread onto the gelatin coating. The cells behave as insulators, limiting the current flow between the electrodes. As more cells attach to the surface, the current flow decreases further. As a result, the impedance, or opposition to the current flow, and potential increase. Eventually, the cells proliferate on the electrodes and form a monolayer.
Treat the SMCs with a calcium ionophore to facilitate calcium ion transport across the cell membranes. The extracellular calcium ion influx activates the contractile apparatus, inducing contraction.
Stimulated SMCs contract and lose cell-cell contact on the electrodes. The resulting reduction in electrode surface coverage decreases the impedance, facilitating real-time quantification of SMC contraction.
Count the cells using an automated cell counter, and seed the SMCs in triplicate at a seeding density of 30,000 cells per well, in 200 microliters of SMC medium in the gelatin-coated ECIS plate. Place the plate with the SMCs into the ECIS 96-well holder in the cell culture incubator.
Double-click on the "ECIS Applied Biophysics software" to open the program, and press the "Setup" button. Check if all the electrodes have contact with the holder, in the left lower panel labeled "Well Configuration." If the electrodes are not properly connected, adjust the plate in the holder before starting the measurement.
Now, select the plate type in the same panel by clicking "Array Type." Next, prepare two pipettes set at 2-microliter and 150-microliter volume. Before starting the stimulation, press "Mark" in the software, and place a comment.
To stimulate the cells, remove the lid, and place it on a sterile surface inside the incubator. Then, induce SMC contraction by pipetting 2 microliters of the ionomycin working solution into each well as quickly as possible.
Once all the cells have been stimulated, mix the medium in the wells using the second pipette.
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