A Plate-Based Assay to Measure Monoamine Release from Brain Slices on Test Drug Exposure
Trascrizione
To induce monoamine release, transfer the tissue samples to each well of a custom-made 48-well efflux chamber, containing 0.5 to 1 milliliter of efflux buffer per well with constant, gentle bubbling, and allow the samples to recover for 30 to 50 minutes at 37 degrees Celsius.
At the end of the equilibration period, move the tissue holder with brain tissue, to wells containing 500 microliters of oxygenated efflux buffer with or without pharmacological agent, tapping the holder on the edge of the well to prevent the minimal transfer of buffer between wells, for a 20-minute incubation at 37 degrees Celsius.
At the end of the incubation, move the holder to a new set of wells containing 500 microliters of efflux buffer with or without the drug of interest, and return the plate to the cell culture incubator, for an additional 20 minutes.
During the second incubation, transfer the solution from the wells from the first incubation into microcentrifuge tubes containing 50 microliters of 1 N perchloric or phosphoric acid on ice, and label the tubes #1.
At the end of the second incubation, move the tissue holder to empty wells with the plate on ice, and transfer the supernatants from the second incubation to new microcentrifuge tubes containing 50 microliters of 1 N perchloric or phosphoric acid, on ice. Label these tubes #2.
When all of the supernatants have been transferred, add 1 milliliter of ice-cold dissection buffer to each well of tissue, and use small tweezers to transfer each tissue sample to a new microcentrifuge tube. Remove the buffer from the sample tubes, and store the tissues at -80 degrees Celsius. Then, transfer the collected incubation solutions into microcentrifuge filter tubes for centrifugation, and place the filtrate on ice, until HPLC analysis.
