Glomax navigator luminometer

Amyloidogenic processing of APP was monitored in HEK293 cells essentially as described using an APP-GAL4 reporter assay that drives GAL4 upstream activator sequence (GAL4-UAS)-dependent expression of firefly luciferase [22 (link)]. Briefly, APP fused in frame at its C-terminus to GAL4 was co-transfected into HEK293 cells with a GAL4-UAS-firely luciferase reporter gene (pFR-Luc), Renilla transfection efficiency control plasmid (pRL-CMV) and either KLC1wt or KLC1S460D. BACE1, α-secretase and γ-secretase cleavage of APP releases the APP intracellular domain fused to GAL4 which translocates to the nucleus to regulate expression of firefly luciferase. Transfection efficiency normalised firefly luciferase signals thus provide a readout for amyloidogenic processing of APP. Cells were transfected in 24 well plates at 70–80% confluence with 0.25 μg each of APP-GAL4, pFR-Luc and either KLC1wt or KLC1S460D, along with 0.025 μg of pRL-CMV Renilla control plasmid. Cells were transfected as described above (see Cell culture and transfection). Luciferase signals were developed 24 h later using a Dual-Glo Luciferase assay kit (Promega) according to the manufacturer’s instructions and quantified using a Promega GloMax Navigator luminometer. Full details of the assay including design and construction of plasmids have been described previously [22 (link)].

Bacterial log-phase cultures were diluted in culture medium/SHU or filtered human urine to an OD₆₀₀ of 0.01 (~10⁷ CFU/ml) and infected with reporter phages at an MOI of 0.1 (E. coli and Klebsiella) or at an MOI of 2 (Enterococcus). Sterile medium spiked with reporter phages alone served as background controls. For E2, E4, K1, and K4, the infection reactions were incubated at 37 °C with shaking (180 rpm) for a total duration of 3 h. For EfS3 and EfS7, the reactions were incubated at 30 °C without shaking for 4 h. For time-course assays, an aliquot of each mixture was taken every 20 min for luminescence measurement in Nunc^TM F96 MicroWell^TM plates (Thermo Fisher) and using a GloMax® navigator luminometer (Promega) with 5 s integration and 2 s delay. Based on manufacturer’s instructions, the buffer-reconstituted NLuc substrate (Nano-Glo Luciferase Assay System; Promega) was added to the infection mixtures at 1:1 ratio (40 µL total). Relative light units (RLUs) were divided by the RLU from phage-only controls and quantified as fold change RLU. All strains used for specificity assays are listed in Table S2.