Microamp 96 well reaction plates

Gene expression assays were performed with a direct lysis microplate assay.²⁵ Cells were washed with 0.9% w/v saline before lysis in 5 mM Tris, 75 mM saline, and 0.05% Triton X‐100 for 5 min. RT‐qPCR reactions were performed with MicroAmp 96‐Well Reaction Plates (4366932, Thermo Fisher Scientific). Thermal cycling was performed with a 5 μL template and 5 μL reverse transcriptase TaqPath 1‐Step RT‐qPCR Master Mix (A15300, Thermo Fisher Scientific) at 50°C for 15 min, enzyme activation 2 min, amplification 95°C for 3 s/60°C for 15 s (Applied Biosystems QuantStudio 5). TaqMan predesigned assays (exon junction spanning) were used, including Hs00389217_m1 (S100B), Hs00188193_m1 (SLC1A3), Hs01102423_m1 (SLC1A2), Hs00909233_m1 (GFAP), Hs02596860_s1 (MT‐RNR2), Hs00173304_m1 (PPARGC1A), Hs01009006_m1 (SIRT1), Hs02758991_g1 (GAPDH), and Hs00427620_m1 (TBP) (4,331,182, Thermo Fisher Scientific). For IL6 activation assays, astrocyte cultures were treated with saline or 100 μg/mL polyinosinic:polycytidylic acid (poly(I:C)) overnight. Data were acquired with an Applied Biosystems Scientific QuantStudio 5 and analyzed with Quantstudio v1.5.1.

Gene expression experiments were conducted with an Applied Biosystems Scientific QuantStudio 5. RT-qPCR reactions were carried out in 10 µL volumes in MicroAmp 96-Well Reaction Plates (4366932, Thermo Fisher Scientific, Waltham, MA, USA). Each target or multiplex combination was prepared with TaqPath One-Step RT-qPCR Master Mix (A15300, Thermo Fisher Scientific, Waltham, MA, USA). Standard thermal cycling conditions were performed with reverse transcriptase at 50 °C for 15 min (1×), enzyme activation at 95 °C for 2 min (1×), and amplification 95 °C for 3 s, followed by 60 °C for 15 s (40×) (Applied Biosystems QuantStudio 5). TaqMan predesigned assays (exon junction spanning) included Hs00174131_m1 IL6, Hs00169585_m1 PPP1R15A, Hs00389217_m1 S100B, Hs00909233_m1 GFAP, Hs00904823_g1 SLC1A3, Hs00220404_m1 SLC17A7, Hs00199577_m1 SYN1, Hs02758991_g1 GAPDH, Hs02800695_m1 HPRT1 (4331182, Thermo Fisher Scientific, Waltham, MA, USA), and Hs00427620_m1 TBP (4448489, Thermo Fisher Scientific, Waltham, MA, USA).