For ethanol-treatment experiments mixed-sex primary astrocyte cultures were grown in 100 mm dishes. At the end of the treatments, cells were washed twice, scraped in PBS, and centrifuge at 200 × g. Cell pellets were re-suspended in deionized water and sonicated 5 times at 30% for 5 s each on ice. Cell homogenates were then centrifuged for 10 min at 250 × g and the supernatant used for plasmin activity determinations using the Plasmin Activity Assay kit (AnaSpec, EGT Group, Fremont, CA) as described by the manufacturer.
For siRNA- and recombinant tPA-treatment experiments, primary astrocytes were grown in 24-well plates. At the end of each treatment, wells were washed 2x with PBS and 150 μl of 1× assay buffer was added to each well. An additional 150 μl of 1× assay buffer containing 150 μM plasmin substrate was then added to each well. Plates were incubated for 1 h at 37 °C in a tissue-culture incubator under an atmosphere of 5% CO2/95% air after which fluorescence intensity was read (Ex/Em = 380 nm/500 nm) using a SpectraMax Gemini EM (Molecular Devices, Sunnyvale, CA, USA). The relative fluorescence units (RFU) measured in each sample was normalized to the protein added to each well as determined by the BCA method.
For siRNA- and recombinant tPA-treatment experiments, primary astrocytes were grown in 24-well plates. At the end of each treatment, wells were washed 2x with PBS and 150 μl of 1× assay buffer was added to each well. An additional 150 μl of 1× assay buffer containing 150 μM plasmin substrate was then added to each well. Plates were incubated for 1 h at 37 °C in a tissue-culture incubator under an atmosphere of 5% CO2/95% air after which fluorescence intensity was read (Ex/Em = 380 nm/500 nm) using a SpectraMax Gemini EM (Molecular Devices, Sunnyvale, CA, USA). The relative fluorescence units (RFU) measured in each sample was normalized to the protein added to each well as determined by the BCA method.