This study investigates the permeability of a blood-brain barrier (BBB) model using fluorescein isothiocyanate (FITC) and FITC-loaded ferritin. The results demonstrate differential transport mechanisms across the BBB, highlighting receptor-mediated internalization.
Begin with a multiwell plate containing an in vitro blood-brain barrier or BBB model.
This model consists of endothelial cells adhered to the upper or blood side and astrocytes on the lower or brain side of a matrix-coated membrane insert.
Add fluorescein isothiocyanate or FITC, a fluorescent dye, to the upper chamber of a control well.
Conversely, add FITC-loaded ferritin, or FITC-ferritin, to the upper chamber of a sample well and then incubate.
In the control well, free FITC remains confined to the upper chamber, with minimal crossing through the BBB.
Meanwhile, in the sample well, FITC-ferritin complexes bind to receptors on the endothelial cells.
This allows receptor-mediated internalization and transport into the lower chamber.
Next, regularly collect medium from the lower chambers of both wells and measure fluorescence intensity.
The control exhibits lower fluorescence, while the sample displays higher fluorescence, confirming the differential BBB permeability for FITC and FITC-ferritin.
To measure the FITC-ferritin flux through the blood-brain barrier system, add FITC-loaded ferritin or free FITC to the upper portion of at least three blood-brain barrier systems for each formulation.
After seven hours in an incubator, withdraw 2 milliliters of medium from the lower chambers of three sample and control inserts. Measure the fluorescence intensity of 500 microliters of the collected samples by spectrofluorometer, and determine the concentration of permeated FITC or FITC-ferritin according to the instructions in the written portion of the protocol.
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