Iddings (Cornell University) for technical assistance during measurement of OCR in MA-10 cells. was performed using a luciferin-luciferase method with an Enliten ATP detection kit (Promega), and luminescence was measured using an Infinite 200Pro reader (Tecan). Measured ATP values were normalized to total protein content for comparison between groups. Measurement of mitochondrial membrane L-Stepholidine potential (among samples. Measurement of ROS Production of ROS and superoxide were directly monitored in live cells by flow cytometry using the total ROS/superoxide detection kit (Enzo Life Science). Cells were grown overnight to 60% density, trypsinized, and washed with PBS. Cells L-Stepholidine were then incubated with fluorescent dyes: oxidative stress detection reagent (green) for total ROS and reactive nitrogen species (RNS), and superoxide detection reagent (orange) for 30 minutes at 37C in the dark. The labeled MA-10 cells from both genotypes were examined using wavelengths 488/520 nm and 550/620 nm in a Gallios flow cytometer (Beckman Coulter). Positive controls (200M pyocyanin treatment) and negative controls L-Stepholidine (5mM N-acetyl-L-cysteine treatment) were used to confirm analytical methods. Mean fluorescence intensity was calculated from data collected from the 2 2 channels, and values were compared between the 2 genotypes using Kaluza Flow Analysis software (Beckman Coulter). Lentiviral expression of TSPO in MA-10 cells cDNA was cloned in a lentiviral vector (pLenti CMV GFP Puro) (34) for expression in mammalian cells. Lentiviral particles were packaged using 293T cells by cotransfecting with helper plasmids encoding gag, pol, and rev, and viral supernatants were collected at 48 and 72 hours. Unmodified pLenti vector was used to make green fluorescent protein (GFP) control viruses. MA-10:and control lentiviruses in medium supplemented with 6 g/mL Polybrene (Sigma) for 24 hours. Infection efficiency was analyzed visualizing GFP fluorescence L-Stepholidine and TSPO expression after 5 days before cells were used for experiments. Gene expression Total RNA was extracted from MA-10 cells and adrenal glands using TRIzol (Life Technologies). Reverse transcription was carried out using Multiscribe reverse transcriptase (Life Technologies). Gene expressions were quantified using a SYBR Green detection method (Affymetrix) with validated primer sequences obtained from PrimerBank database (35). Primer specificity and amplification efficiency were confirmed and sequences are provided in Supplemental Table 1. All expression data were normalized to internal control genes, TATA-binding protein (test; comparisons for more than 2 groups were performed using ANOVA and post hoc Tukey’s test (< .05 was considered significant). All analyses were performed using Prism 5 (GraphPad). Data are represented as mean SEM, and replicates are as indicated in figure legends. Results TSPO deletion does not affect mitochondrial respiration in MA-10 cells Complete absence of TSPO in the 2 2 MA-10:(n = 5C6/group). I, Total ATP levels were not different between MA-10:and ATP production in MA-10 cells Assessment of using the membrane potential-dependent probe TMRM, normalized to Mitotracker Green showed that MA-10:(Figure 1H). Provided that oxygen consumption and were unchanged, ATP production was not expected to be IL17RA different between MA-10:were significantly up-regulated in MA-10:and peroxisomal -oxidation gene between the 2 genotypes. Expression levels of key genes in lipogenesis pathway: were not different between MA-10:was significantly lower in one clone MA-10:< .05). Expression levels of crucial genes involved in fatty acid metabolism indicated that MA-10:< .05. ROS and UCP2 are up-regulated in MA-10:gene expression and.