Taqman mouse primers

Gene expression for Tspo, Cybb, Cyba, VDAC1, and GAPDH was performed. RNA was isolated from primary mouse microglial cells treated with vehicle or 100 ng/mL LPS for 18 h by using RNAqueous Micro Kit (AM1931, Invitrogen, Carlsbad, CA). RNA content was measured using NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Reverse transcription of RNA was performed with High-Capacity RNA-to-cDNA kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). qRT-PCR was performed using 1 μl of cDNA diluted to 20 ng/μl, TaqMan multiplex master mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) and TaqMan mouse primers (Thermo Fisher Scientific, Waltham, MA) in a final reaction of volume of 10 μl. Primers used included Tspo (Mm00437828_m1-FAM-MGB); Cybb (Mm01287743_m1-FAM-MGB); Cyba (Mm00514478_m1-FAM-MGB); Vdac1 (Mm00834272_m1-FAM-MGB); and Gapdh (Mm999999915_g1-VIC-MGB). The results were evaluated using QuantStudio Real-Time PCR Software v1.3 (Thermo Fisher Scientific, Waltham, MA). Amplification specificity was confirmed by melting curve analysis, and the quantification was carried out using the ΔΔCt method [45 (link)]. All samples were normalized to Gapdh. Data from six independent experiments were run on the same plate for each gene. All individual samples were run in triplicate.

Mouse macrophage Raw264.7 cells were treated with vehicle (0.1% DMSO) or neratinib in triplicates at different concentrations for 2 h, followed by 100 ng/mL LPS stimulation for 4 h. Cells were washed and RNA isolated by using RNeasy Mini Kit (Qiagen). One microgram of cDNA of each treatment sample was synthesized by using SuperScript III First-Strand Synthesis (Invitrogen). Taqman mouse primers were purchased from ThermoFisher: TNFα (Catalog No. Mm00443258_m1), IL-6 (Catalog No. Mm00446190_m1), IL-1β (Catalog No. Mm00434228_m1), and GAPDH as endogenous housekeeping control (Catalog No. Mm99999915_g1). The qPCR reaction was set up for TNFα, IL-6, IL-1β, and GAPDH individually, with technical triplicates for each gene per treatment sample and performed by the Applied Biosystems ViiA 7 real-time PCR system. The ∆∆CT method was used to analyze the relative changes in gene expression.