Fluostar omega plate reader

Time-course turbidity assays were performed for wild-type phage A006 and A006::egfp_cps to demonstrate that the lysis kinetics of both phages are comparable. To this end, mid-exponential L. monocytogenes Rev2 cells expressing chromosomally integrated RFP were pelleted at 12,000 × g for 4 min, resuspended in DM3_Φ and adjusted to OD₆₀₀ of 0.1 (≈10⁸ bacteria per ml). Diluted culture (190 µl) was infected with 10 µl of A006 or A006::egfp_cps phage lysate (10¹⁰ p.f.u. ml⁻¹(plaque forming units)). Turbidity was monitored at 2 min intervals at 30 °C in flat-bottom 96-well plates using a FLUOstar OMEGA plate reader (BMG LABTECH). Plates were agitated before each measurement. Identical infection conditions were used for fluorescence time-course assays. Fluorescence intensities were measured in black-walled 96-well plates with a FLUOstar OMEGA plate reader (BMG LABTECH) at 485 nm excitation wavelength with a 520 nm emission filter. Fluorescence time-course assays were background corrected by subtraction of controls (bacteria+ phage A006). All data were acquired using OMEGA software v5.10 in three independent experiments.

The cytotoxicity of various liposomal formulations of ATO was determined by the MTT assay, as described previously [31 (link)]. Briefly, the experiment was set up in a 96-well plate, where the toxicity of control empty liposomes was investigated by taking an initial starting amount containing 0.5 mM of phospholipid concentration and diluting it further in a 1:10 ratio to a further six wells. The ATO encapsulating liposomes contained 30 µM of ATO in the initial sample, which was further diluted to a 1:6 ratio down to six wells. The wells were seeded with HeLa, HT-3, CRL-1790, and HK at 0.6 million cells per mL and incubated at 37 °C in the humidified incubation chamber for 24 h, 48 h and 72 h. After each time interval, the spent media was removed carefully and 50 µL of MTT solution was added into each well. After 30 min incubation at 37 °C, with 95% O₂ and 5% CO₂, MTT reagent was removed carefully from each well, 100 μL propanol was added to dissolve the crystals, and it was incubated at 37 °C for at least 30 min. The absorbance of this coloured solution was quantified by measuring at a wavelength of 570 nm, using a BMG LabTech FLUOstar Omega Plate Reader (Bucks, UK).

Patient serum was initially diluted 1:10 and then serially diluted 1:5.
SARS-CoV-2–pseudotyped virus was added to each serum dilution and incubated
at 37 °C for 1 hour. The serum-pseudotyped virus mixture was added to
HEK-293T cells stably transfected to express ACE2 and incubated at 37 °C for
48 hours. The Bright-Glo Lucifearase Assay System (Promega) was used to lyse
cells and produce the luciferase readout, which was measured with a FLUOstar
Omega plate reader (BMG Labtech). Relative luminescence units were
normalized to pseudovirus and media-alone readouts. IC₅₀ values
were calculated by nonlinear regression (Prism; GraphPad).

Salmonella strains were grown in triplicate in the appropriate media and samples of 150 μl of each culture were used to measure luminescence and OD₆₀₀, in 96-well white clear bottom plates using a FLUOstar Omega plate reader (BMG LABTECH). To measure the luminescence of intracellular bacteria, RAW264.7 macrophages were plated in 96-well white clear bottom plates at 3 x 10⁴ cells per well, and were infected 24 h later with non-invasive bacteria (grown in LB for 24 h at 37°C with shaking) at a multiplicity of infection of 500. The cell culture was washed twice with PBS 30 min p.i., overlaid with DMEM containing 100 μg/ml gentamicin, and incubated for 1.5 h. The culture was then washed twice with PBS, covered with DMEM with 16 μg/ml gentamicin and incubated for 6 additional h. Luminescence was measured at 2, 4 and 8 h p.i. and the colony forming units per well were calculated after incubation with 1% Triton X-100 in PBS for 10 min at 37°C to release bacteria, plating appropriate dilutions in LB with Ap, and counting colonies after 24 h incubation at 37°C.