Evans blue dye as an indicator of albumin permeability across a brain endothelial cell monolayer in vitro

Neuroreport. 2021 Aug 4;32(11):957-964. doi: 10.1097/WNR.0000000000001690.


An increase in the brain endothelial (BEnd) cell permeability of blood albumin is often seen as an early sign of blood-brain barrier (BBB) disruption and can precede increases in the BEnd permeability of small molecules and other plasma proteins in the course of brain disease. Therefore, Evans blue dye (EBD), an albumin-binding fluorescent tracer that is simple to detect and quantify, has been widely utilized for studying BEnd permeability during BBB disruption. Here, we investigated whether EBD is a suitable indicator of albumin permeability across mouse BEnd cell monolayers, alone or cocultured with mouse cortical astrocytes, in an in-vitro permeability assay; given the strong affinity of EBD for albumin, we further asked whether EBD can affect albumin permeability and vice versa. Albumin and EBD readily crossed membrane cell culture inserts with pore diameters of no less than 1 µm in the absence of a cellular barrier, and their permeability was substantially reduced when the membranes were overlaid with a monolayer of BEnd cells. In line with albumin binding, the BEnd permeability of EBD was substantially reduced by the presence of albumin. While EBD at an EBD-to-albumin ratio similar to those typically used in in vivo BBB experiments had little effect on the BEnd permeability of albumin, a much higher concentration of EBD augmented the BEnd permeability of albumin. In conclusion, we investigated the use of EBD as an indicator of albumin permeability in vitro, explored some of its drawbacks and further demonstrated that EBD at the concentration used in vivo does not affect albumin permeability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood-Brain Barrier / drug effects
  • Blood-Brain Barrier / metabolism*
  • Brain / drug effects
  • Brain / metabolism
  • Capillary Permeability / drug effects
  • Capillary Permeability / physiology*
  • Cell Line
  • Cells, Cultured
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism*
  • Evans Blue / administration & dosage
  • Evans Blue / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Serum Albumin, Bovine / administration & dosage
  • Serum Albumin, Bovine / metabolism*


  • Serum Albumin, Bovine
  • Evans Blue