Envision 2105 multimode plate reader

The ATP cell content was measured using Cell Titer Glo (#G7571, Promega, Charbonnières Les Bains, France). In summary, the cells were seeded at 2000 cells/well in three replicates in 384-well ViewPlates (Perking Elmer). After an overnight, oligomycin A (Sigma-Aldrich) and sodium iodoacetate (Sigma-Aldrich) were added, either both or in combination. Following a 1 h incubation, the cell confluence was assessed with IncuCyte for further normalization and 40 μL of Cell Titer Glo reaction mix was added to each well, reaching a final volume of 80 μL. The plates were then analyzed for luminescence with an EnVision 2105 Multimode Plate Reader (Perkin Elmer). By comparing the different conditions, the global ATP and the percentages of both glycolytic and mitochondrial ATP were determined.

The 96-wells assay plate (white opaque) containing compounds were prepared by adding 1 μL of compound dissolved in DMSO per well in triplicates. The transfected cells cultured in 6 well plates were trypisinized and re-suspended in assay media (phenol-free complete DMEM media containing 10% charcoal stripped fetal bovine serum). Cells (10,000 cells in 100 μL per well) were added to the compound plate per well. After 24 h, media was replaced in each well with same fresh media and 1 µL of desired compound dissolved in DMSO or DMSO alone as control was added to each well to give the final concentration 0.1, 1 or 10 μM. The cells were then continued to be cultured for another 24 h prior assaying the luciferase activity using Dual-Glo^® Luciferase Assay System (Promega). One hundred μL of the Dual-Glo^® Luciferase buffer containing luciferase substrate were added to measure firefly luciferase (the promoter activity) and read on a Envision 2105 Multimode Plate Reader (PerkinElmer, American Fork, UT, USA). Then 100 μL of Stop-Glo buffer containing Stop-Glo substrate to measure renilla and plate were read again in the Perkin Elmer Envision system. The reporter induction was calculated by dividing the luciferease signal on the renilla signal.

Determination of cytokine levels from primary moDCs was performed using ELISA. Supernatants were collected in the case of immature moDCs on day 5 and for the matured cells on day 6. IL-6, -8, -10, -12, and TNFα ELISA kits were all from BD-Biosciences, and determination was performed according to the manufacturer’s instructions. The cytokine levels were measured by EnVision 2105 Multimode Plate Reader (Perkin Elmer). The concentration of the cytokines were then calculated with cubic logistic model (52 (link)) using GraphPad Prism 9.1.2 for Windows (GraphPad Software Inc., La Jolla, CA, USA).

Active MMP-8 concentrations were determined in GCF samples by IFMA, as described by Hemmilä [23 (link)]. Briefly, IFMA is based on anti-MMP-8 recognition by the monoclonal MMP-8 specific antibodies 8708 and 8706 (Oy Medix Biochemica Ab, Espoo, Finland), as a catching antibody and a tracer antibody, respectively. The tracing antibody was labeled with europium chelate. The samples were diluted in assay buffer, and after adding an enhancement solution, fluorescence was measured using an EnVision 2105 Multimode Plate Reader (PerkinElmer, Turku, Finland). The specificity of the monoclonal antibodies against MMP-8 corresponded to that of polyclonal MMP-8. The detection limit for the assay is 0.08 ng/mL.

Ub-Rhodamine 110 (Cat# SBB-PS0001, South Bay Bio) activity assays were determined using 1 nM of purified enzyme with increasing concentration of substrate (Ub-Rho110) in 10 µl reaction buffer (50 mM HEPES pH 7.5, 50 mM KCl, 5% glycerol, 5 mM MgCl2, 5 mM DTT, 0.1 mg/ml BSA, and 0.005% Tween-20). Experiments were performed at 37 °C in black 384-well non-binding surface low flange plates (Corning) and monitored in an EnVision 2105 Multimode Plate Reader (PerkinElmer) using 350 nm and 450 nm excitation and emission wavelengths, respectively. Measurements were taken every 60 s for 90 min.