Escherichia coli k 12 bioparticles

To assess phagocytosis, microglial cells cultured in 6-well plates were digested using 0.25% trypsin-EDTA and re-seeded to a 96-well plate at a concentration of 3.0 × 10⁴ cells/well. Then, the cells were incubated with fluorescein-labeled Escherichia coli K-12 BioParticles (Invitrogen) for 2 h at 37 °C. The cells were rinsed with 0.25 mg/ml trypan blue to quench extracellular fluorescence. Intracellular fluorescence was read using a fluorescence microplate reader setup with excitation at 480 nm and emission at 520 nm. The experiments were performed with five replicates per condition and repeated four times.

Cell migration assay kit (Chemicon, Billerica, MA) was used to analyze microglial migration. Microglia suspension was loaded into the culture well inserts (membrane pore size of 5 μm) and placed in 24-well plates containing serum-free DMEM/F12 media in the presence or absence of the conditional media from OGD-treated endothelial cells or astrocytes. After incubating for 4 h, the migrated cells were detached from the bottom of the insert and lysed and stained using the CyQUANT^® GR Dye. The samples were read using a fluorescence microplate reader set up at 480 nm excitation and 520 nm emission. Migrated cell number was determined by running a fluorescent cell dose curve. To assess phagocytosis, microglial cells were seeded to a 96-well plate at a concentration of 1.0 × 10⁵ cells/well and incubated with or without the conditional media from OGD-treated endothelial cells or astrocytes for 24 h. Then, the cells were incubated with fluorescein-labeled Escherichia coli K-12 BioParticles (Invitrogen, Grand Island, NY) for 2 h at 37 °C. Cells were rinsed with 0.25 mg/ml trypan blue to quench extracellular fluorescence. Intracellular fluorescence was read using a fluorescence microplate reader set up with excitation at 480 nm and emission at 520 nm. The experiments were performed with five replicates per condition and repeated a minimum of three times.