Axio observer z1 motorized fl inverted microscope

PC-3 cells were seeded (20 000 cells/500 μL RPMI/well) in a 24-well plate overnight. The cells were washed twice in PBS and incubated for 8 h in triplicates with 5.0 μM PS, 5.0 μM PS-PhMV, or the corresponding concentration of unloaded VLPs (236.70 μM or ~0.19 mg/mL). After washing twice in PBS, 500 μL of RPMI medium was added. Photodynamic therapy was then applied using a white light source (Phillips Silhouette High Output F39T5/841 HO, Alto collection, ~10 mW cm⁻²) for 30 min (18.1 J cm⁻² at 430 nm), and cells were incubated for a further 48 h in the dark. Cell viability was determined using a LIVE/DEAD assay for mammalian cells (Thermo Fisher Scientific) following the manufacturer’s procedures for cell staining, and cells were observed under a Zeiss Axio Observer Z1 motorized FL inverted microscope.

Cell viability was assayed using MTT and LIVE/DEAD assays. Confluent cells were removed using 0.05% (w/v) trypsin-EDTA, added to 96-well plates (100 µL/well, 2 × 10⁴ cells/mL), and grown overnight. Native TMV, drug loaded _Zn-EpPorTMV, or free Zn-EpPor was added to cells using 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 5.0 µM Zn-EpPor; cells were incubated for 8 h at 37 °C and 5% CO₂. Assays were done in triplicate and repeated at least twice. Following incubation, cells were washed twice to remove unbound drug and drug carriers, and then 100 µL of medium was added. Samples were illuminated in a rectangle (10.5 cm × 11 cm) under white light from a Vivitek D950HD projector (~10 mW cm⁻² at 430 nm) for 30 min (18.1 J cm⁻² at 430 nm). Control samples were kept in the dark for 30 min. After illumination, plates were incubated at 37 °C and 5% CO₂ for 48 h. Cell viability was assessed using an MTT cell proliferation assay (ATCC); the procedure was as per manufacturer’s recommendation. Alternatively, cell viability and cytotoxicity was determined using the live life in LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (ThermoFisher). The staining procedure was as per manufacturer’s recommendation. Plates were imaged on a Zeiss Axio Observer Z1 motorized FL inverted microscope. Images were analyzed for percentage cell viability using ImageJ 1.47d (http://imagej.nih.gov/ij).