This article describes a method for recording the resting membrane potential of arteriolar endothelial cells harvested from mouse brains. The technique involves the use of calcium-sensitive fluorescent dyes to measure intracellular calcium levels and assess the functional status of the endothelial tube.
Secure an arteriolar endothelial tube harvested from a mouse brain in a chamber with a continuous buffer flow.
Place the chamber in a recording setup.
Introduce a calcium-sensitive fluorescent dye. Incubate to allow cellular uptake of the dye.
Wash to remove any non-internalized dye.
Insert an electrode into an endothelial cell and record the resting membrane potential.
Raise the bath temperature to a physiological temperature to facilitate intracellular calcium binding to the dye.
Excite the dye and measure fluorescence to quantify baseline cellular calcium levels.
Introduce a drug that binds to specific G-protein-coupled receptors on the endothelial cells, initiating a signaling cascade.
This cascade releases calcium ions from the endoplasmic reticulum into the cytoplasm, enhancing calcium-dye binding and fluorescence.
Elevated cytoplasmic calcium levels also activate potassium channels, facilitating potassium ion efflux and decreasing membrane potential.
Record the data.
Increased fluorescence and decreased membrane potential indicate a functional endothelial tube.
To measure endothelial membrane potential, while viewing through the four times objective, carefully position the sharp electrode tip just over a cell of the arterial endothelial tube into the flowing physiological salt solution with a micromanipulator. Gradually, increase magnification to 400 times and reposition the electrode tip as needed. Using the micromanipulator, gently insert the tip of a sharp electrode into one of the cells of the endothelial tube, and start recording VM using an electrometer.
Once the endothelial resting VM is stable from minus 30 to minus 40 millivolts, apply the desired pharmacological agents per experimental objective. Load the endothelial tube with the fluorescence tracker for the plasma membrane or desired organelle at 37 degrees Celsius for 15 to 30 minutes. Wash the cells with fresh superfusion physiological salt solution, and image live cells under the microscope at the excitation wavelength of the respective dyes.
繁體中文
