Envision multilabel plate reader

For the cell membrane studies, S. aureus in mid-log growth phase was centrifuged (1000g, 3 min), washed, and resuspended in PBS, and then it was exposed to 405-nm irradiation or control conditions (maintained in the dark). Additionally, untreated bacteria were exposed to 0.01 % benzethonium chloride (BCl; cell membrane disruption, positive control) for 1 h. Then, propidium iodide (PI; at a final concentration of 5 μg/ml) was placed in each sample, and the cells were incubated for an additional 30 min in the dark at room temperature. Following staining, the bacteria were centrifuged, washed, and resuspended in 1-ml PBS. The fluorescence of the samples was read using an EnVision Multilabel Plate Reader (PerkinElmer) with 488/570 nm excitation and emission filters. Additionally, the samples were stained with SYTOX Green (Molecular Probes) at a final concentration of 5 μM for 10 min at room temperature. Finally, the fluorescence signal of DNA-bound SYTOX Green was measured using an EnVision Multilabel Plate Reader (Perkin Elmer) with 504/523 nm excitation and emission filters. The experiments were performed three times and analyzed statistically (Connell et al. 2013 (link)).

For cell membrane studies, S. aureus in mid-log growth phase, centrifuged (1000 × g, 3 min), washed, and re-suspended in PBS, was exposed to APDT treatment or control conditions (treated with fullerene in dark, treated only with light and kept in dark). Additionally, non-treated bacteria were exposed to 0.01 % benzethonium chloride (BCl; cell membrane disruption positive control) for 1 h. Next, propidium iodide (PI; at a final concentration of 5 μg/ml) was placed in each sample, and the cells were incubated for an additional 30 min in the dark at room temperature. Following staining, the bacteria were centrifuged, washed, and re-suspended in 1 ml PBS. Sample fluorescence was read using an EnVision Multilabel Plate Reader (PerkinElmer) with 488/570 nm excitation and emission filters. Additionally, samples were stained with SYTOX Green (Molecular Probes) at the final concentration of 5 μM for 10 min in room temperature. Next, measuring of fluorescence signal of DNA-bound SYTOX Green was performed using an EnVision Multilabel Plate Reader (PerkinElmer) with 504/523 nm excitation and emission filters. Experiments were performed three times and analyzed statistically (Connell et al. 2013 (link)).

The levels of IL-4, IL-5, IL-13, TGF-β1 (Invitrogen) or collagen I α1 (Bio-Techne) in the supernatants after the cell treatments were assayed with ELISA kits, according to the manufacturer’s instructions. The concentrations of PGD₂ and LTE₄ in the supernatants were measured with a PGD₂–MOX enzyme immunoassay kit and LTE₄ enzyme immunoassay kit (Cayman Chemical), respectively, according to the manufacturer’s instructions. The results were measured in an EnVision Multilabel Plate Reader (PerkinElmer).

Cell viability was determined by a resazurin-based assay as described previously [22 (link)]. Briefly, 5 × 10³ cells/well were seeded into 96-well plates (Corning Inc., Corning, NY, USA) in 180 µL of RPMI 1640 medium and incubated overnight at 37 °C with 5% CO₂. Next day, cells were treated with rilpivirine after serially diluted (1:2 dilution for final concentrations ranging from 0.625 µM to 40 µM) or DMSO (0.1%) for specified time periods (24, 48, or 72 h). At the end of the treatment period, 20 µL of resazurin (0.1 mg/mL in PBS) was added per well and incubated for further 4 h. Fluorescence intensities were recorded at 570 nm (excitation)/585 nm (emission) using an EnVision^® multilabel plate reader (PerkinElmer, MA, USA). Drug concentrations required for cell viability reduction by 50% (GI₅₀) compared with untreated (DMSO vehicle-treated only) control were determined by a non-linear regression model using GraphPad Prism software version 7.02 (La Jolla, CA, USA).

Caspase-3/7 activity was measured after incubation of the cells with the test compounds for a specified time point using an Apo-ONE homogeneous caspase-3/7 kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The signal was recorded by the EnVision multi-label plate reader (PerkinElmer, Beaconsfield, UK).