In Vitro Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules

First, collect 100 microliters of the medium from both the blood and the brain sides of the BBTB mimic and transfer each to a separate flat-bottomed, black 96-well plate for subsequent fluorescence measurements.

Next, prepare a 50-micromolar solution of sodium fluorescein in EBM minus, making sure to prepare 2.5 milliliters per well. Prewarm the solution to 37 degrees Celsius and replace the media from the blood side of the inserts with media containing this sodium fluorescein solution. Start a timer as soon as the medium is replaced.

Carefully collect 100 microliters of media from both the blood and brain sides of the inserts at 5 minutes, 30 minutes, 60 minutes, and 120 minutes. Transfer each sample to separate wells of the appropriate black 96-well plate. Use a plate reader to quantify the fluorescence from the collected samples.