Transfection of primary astrocytes

Astrocytes were transfected in 6-well plates with SMARTpool siRNAs (Dharmacon) against fibulin-2 or non-targeted control using X-tremeGENE^™ siRNA transfection reagent (Millipore Sigma) according to the manufacturer’s instructions. Briefly, astrocytes were grown at ~80%–90% confluency in 6-well plates. Before transfection, media was changed to antibiotic-free glia culture media. For transfection of each well, 2.5 μL of 100 μM siRNAs was diluted in 125 μL of serum free MEM in one tube and 25 μL of transfection reagent was added to 100 μL of serum free MEM in another tube. The resulting mixture from both tubes was combined immediately, incubated at room temperature for 20 min and added dropwise to the cells. 48 h post transfection, the cells were washed three times with HBSS and conditioned with neurobasal media for 24 h. Conditioned media was subjected to differential ultracentrifugation for isolation of purified SEVs.