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Impact of Oxygen-Glucose Deprivation and Reoxygenation on Cell-Cell Interactions
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Take a chambered slide with a monolayer culture of rat brain microvascular endothelial cells, or RBMECs.
The cells are connected by tight junction proteins linked to the actin cytoskeleton, mimicking the endothelial layer of the blood-brain barrier.
Replace the medium with a deoxygenated glucose-free medium, then place the slide in a hypoxia chamber.
The unavailability of oxygen and glucose inhibits ATP synthesis. The remaining ATP is broken down into nucleotide derivatives.
ATP depletion hinders calcium pumps, elevating intracellular calcium that activates oxidoreductase enzymes.
Next, replenish with an oxygenated medium, then place the slide in an oxygenated incubator. The activated oxidoreductases utilize the reintroduced oxygen to degrade the nucleotide derivatives, generating reactive oxygen species or ROS.
ROS disrupts the interactions between tight junction proteins and induces signaling pathways that remodel the actin cytoskeleton into stress fibers, weakening cell-cell interactions.
The monolayer with disrupted intercellular interactions is ready for analysis.
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