5.3. NFAT luciferase assays

Primary astrocytes were prepared as described above, except an NFAT luciferase adenovirus (Ad‐Luc) was added to the serum‐free medium so that the cells were infected with 50 MOI of virus per our previous studies (Furman et al., 2010; Sama et al., 2008). Inhibitors and activators were added as above, using the indicated concentrations of Q134R for dose‐inhibition curves. Cells were maintained in NFAT stimulatory agents for 4–5 hrs in serum‐free medium, then harvested for luciferase assays by washing cultures twice with Ca²⁺/Mg²⁺‐free phosphate‐buffered saline, then adding M‐PER Mammalian Protein Extraction Reagent (Thermo Scientific, Waltham, Massachusetts) to cells and immediately freezing overnight at −20℃. The next day, extracts were thawed and collected and used immediately in luciferase assays as per manufacturer's directions (ThermoFisher, Extended‐Glow Luciferase Reporter Gene Assay System, Catalog number T1033).