PMC

Primary mouse microglia were incubated for 18–24 hrs with either vehicle (basal), TNFα (5 ng/ml), IFNγ (100 Ui/ml), TNFα plus IFNγ, or TGFβ2 (1 ng/ml). Supernatant aliquots were recovered and amounts of (A) IL-1α, (B) IL-6 and (C) RANTES determined by Luminex beads. Values are mean±SEM of six measurements (i.e. three independent experiments, each performed in duplicate). **p<0.01; *p<0.05, significantly different from basal, as determined by ANOVA followed by Dunnett's.

(A) Representative specific binding of [³H]-PK 11195 to DBT cell homogenates (average Bmax and Kd are in ). (B) Competition of specific [³H]-PK 11195 binding to DBT cell homogenates by increasing concentrations of PK 11195 (dotted line) or NIR-conPK (solid line). Ki and EC₅₀ values for PK 11195 competition were 41 and 93 nM respectively, and calculated using three independent experiments, each performed in triplicate. Ki value for NIR-conPK could not be reliably calculated, indicating that this MI agent does not bind to TSPO. (C) Competition of specific [³H]-PK 11195 binding to DBT cell homogenates by increasing concentrations of 6T (dotted line) and NIR-6T (solid line). Ki and EC₅₀ values for 6T were 3.6 and 114 nM respectively and for NIR-6T 400 and 412 nM and were calculated using three independent experiments, each performed in triplicate.

(A, B) Kinetics of the total binding when labeling intact DBT cells with 100 nM of either NIR-conPK (A) or NIR-6T (B). (C, D) Ability of PK 11195 and 6T to compete for the binding of (C) NIR-conPK and (D) NIR-6T in intact DBT cells. DBT cells grown in 96 well plate were pre-incubated for 15 min with either vehicle (media with DMSO, i.e. total), PK 11195 (PK, 10 µM) or 6T (DAA, 10 µM), and then incubated with either (A) NIR-conPK (100 nM) or (B) NIR-6T (100 nM) for one hour. Cells were then rinsed and RFU at 800 nm read with an LI-COR Odyssey® Infrared Imaging system. Values are mean±SEM of nine measurements (i.e. three independent experiments, each performed in triplicate). *p<0.01, significantly different from total, as determined by ANOVA followed by Dunnett's. (E, F) Kd of NIR-conPK when using either (E) PK 11195 (10 µM) or (F) DAA1106 (10 µM) to determine non-specific binding. DBT cells grown in a 96 well plate were pre-incubated for 15 min with either media alone (i.e. total), or addition of PK 11195 or DAA1106, and then incubated with increasing concentration of NIR-conPK for one hour. Cells were then rinsed and RFU at 800 nm read with an LI-COR Odyssey® Infrared Imaging system. Values are mean±SEM of nine measurements (i.e. three independent experiments performed in triplicate). Solid line represents specific binding; dotted line with square shows total binding and dotted line with triangle shows non-specific binding.

(A, C) Primary mouse microglia and DBT (B, D) cells grown in 96 well plate were incubated for 18–24 hrs with either cell culture media (basal), TNFα (5 ng/ml), IFNγ (100 Ui/ml), TNFα plus IFNγ, or TGFβ2 (1 µg/ml). Cells then were pre-incubated for 15 min with either cell culture media (total binding) or DAA1106 (10 µM, non-specific binding), and then incubated with NIR-conPK for one hour. Cells were rinsed and RFU at 800 nm read with an LI-COR Odyssey® Infrared Imaging system. Specific binding was calculated by subtracting values of non-specific binding to total binding. Data are mean±SEM of 15 measurements (i.e. five independent experiments performed in triplicate). (C, D) Average optical density and representative western blot (below the graph) of TSPO expression in cytokine-treated primary microglia (C) or DBT (D) cells. Band corresponding to TSPO migrated at 18 kDa, as previously described .