Victor3v 1420 multilabel reader

Twenty-four hours after seeding the cells, Aβ_1–42 was diluted in PBS to a concentration of 30 μm, aggregated with or without p-FTAA (1.5 mm stock solution) and diluted to a concentration of 30, 3, or 0.3 μm. The solution was incubated for up to 5 h at 37 °C. At the end of the aggregation period, each sample was diluted in serum-free medium to a final dilution of 1:10 (3 μm Aβ_1–42) and added to the cells. After 72 h of exposure, the cells were visualized for morphological changes and photographed using a Nikon TMS-F inverted phase contrast microscope (Nikon Instruments Inc.) equipped with an Olympus Altra20 camera using the analySIS getIT v.5 software (Olympus Soft Imaging Solutions GmbH). Furthermore, cell viability was determined using the Cell viability assay Kit II (XTT assay; Roche Diagnostics GmbH) according to the manufacturer's instructions. The absorbance at 450 nm and 750 nm was measured after 16 h using a Victor3V 1420 multilabel reader (PerkinElmer). As controls, serum-free medium with 5% (v/v) diluents or serum-free medium with 5% (v/v) diluents with 30, 3, or 0.3 μm p-FTAA were used.

AGS cells, a human adenocarcinoma epithelial cell line (ATCC CRL 1739), were grown in RPMI-1650 medium with 10% fetal bovine serum (FBS) in tissue-culture flasks. For the infection assay, cells were seeded in 24-well plates (Orange Scientific) and cultured for 1–2 days to reach 60–80% confluence. Before the infection, the wasted medium was replaced with fresh RPMI-1650 with 5% FBS conditioned in the bacterial incubator (9% CO₂, 91% air atmosphere, and 95% humidity). Cells were infected with G27 Pcag-lux strains at a multiplicity of infection (MOI) of 5, while other 24-well plates filled with medium but without AGS cells were infected with the same amount of bacterial culture and used as control sample. The plates were placed inside the bacterial incubator and luminescence was measured at regular time intervals with Victor3V (1420) multilabel reader (Perkin Elmer), with bottom trail pre heated at 37°C. Luminescence was measured with an integration time of 2 seconds (normal aperture) in the absence of optical filters. The luminescence values of wells filled with plain growth medium were used as blank control and subtracted from the values of the experimental samples. Each infection assay was performed in quadruplicate and the assay was repeated in four independent biological replicates. average values and standard deviations were calculated.