Human brain microvascular ECs were obtained from Neuromics (Minneapolis, MN, USA; catalog number: #HEC02). Cells were cultured in a standard humidified atmosphere (37 °C) containing 5% CO2, as we described [58 (link)]. In some experiments, cells were treated with metformin (0.1, 0.5, and 1 mM), adding glucose at 5 or 25 mM, or mannitol 20 mM as osmotic control, for 48 h.
The endothelial permeability assay was performed as we described and validated [58 (link),59 (link)], using fibronectin-coated transwell filters (Corning Inc., Corning, NY, USA).
Levels of mitochondrial and cellular reactive oxygen species (ROS) were assessed using MitoSOX and CM-H2DCFDA dyes, respectively, as we previously described [60 (link),61 (link)].
Cell death was evaluated using a Caspase-Glo® 3/7 assay (Promega, Madison, WI, USA; catalog number: #G6321), as we described [62 (link)]. All reagents were from Millipore-Sigma (Burlington, MA, USA), unless otherwise stated. All experiments were conducted at least in triplicate by blinded investigators.
The endothelial permeability assay was performed as we described and validated [58 (link),59 (link)], using fibronectin-coated transwell filters (Corning Inc., Corning, NY, USA).
Levels of mitochondrial and cellular reactive oxygen species (ROS) were assessed using MitoSOX and CM-H2DCFDA dyes, respectively, as we previously described [60 (link),61 (link)].
Cell death was evaluated using a Caspase-Glo® 3/7 assay (Promega, Madison, WI, USA; catalog number: #G6321), as we described [62 (link)]. All reagents were from Millipore-Sigma (Burlington, MA, USA), unless otherwise stated. All experiments were conducted at least in triplicate by blinded investigators.