Wallac victor 2 multilabel plate reader

ATP levels were measured in rVλ6 monomer or fibril-treated cells as follows. Cells were seeded at 2 x 10⁴/cm² in a tissue culture-treated opaque-walled black 96-well plate (BD Biosciences, Franklin Lakes, NJ) and grown for 24 h before changing to serum-free DMEM:F12 and treating with rV_Lλ6Wil proteins. At the end of the exposure period, ATP was detected using the Promega CellTiter-Glo Luminescent Cell Viability Assay. The microplates were equilibrated to room temperature and 100 μL of the CellTiter-Glo reagent was added to each well; after 2 min of gentle shaking followed by a 10 min incubation at room temperature, luminescence intensity was measured using a Wallac Victor 2 Multilabel plate reader (Perkin Elmer, Waltham, MA).

Production of intracellular reactive oxygen species was measured using DCFH-DA, a redox-sensitive fluorophore [37 (link)]. After exposing AC10 cardiomyocytes to 1 μM rVλ6Wil fibrils for either 2 or 20 h in 12 well tissue culture-treated plates, cells were washed and incubated with 50 μM DCFH-DA for 45 min at 37°C. The dye solution was replaced with HBSS and cells were incubated for an additional 30 min at 37°C. For a positive control, DCFH-DA-treated AC10 cells in HBSS were exposed to serial dilutions of 30% H₂O₂ for 30 minutes at 37°C. For fluorescence quantitation, the HBSS was replaced with 200 μL per well 90% DMSO/10% PBS and plates were shaken in the dark for 10 min [38 (link)]. The contents of each of the 12 wells was transferred to a separate well in an opaque-walled black 96-well microtiter plate and the fluorescence at 535 nm emission (excitation = 488 nm) was measured using a Wallac Victor² Multilabel plate reader (Perkin Elmer).