Luminescence and fluorescence assays
Luminescence and fluorescence assays were performed on mid-log grown
cells, normalised to the lowest recorded optical density at 750 nm, in
96-well microplate format using a FLUOstar Omega Microplate Reader (BMG
Labtech, Buckinghamshire, UK). Absorbance measurements
(OD750) were performed in Greiner CELLSTAR round bottom
clear well microplates (Sigma-Aldrich; M9311), fluorescence measurements
in black Greiner microplates (Greiner Bio-One; 655900), and luminescence
measurements in white Eppendorf microplates (Thermo Fisher Scientific;
15294516).
Luminescence analysis of LucCP expression was performed using the
Steady-Glo Luciferase Assay System (Promega UK Ltd, Southampton, UK).
100 μL of normalized cells were mixed with 100 μL of the Steady-Glo
assay reagent in triplicate. After a 5 min incubation period, the
luminescence signal was measured over the entire visible light range to
maximize the signal to noise ratio. Luminescence was measured and
expressed as relative luminescence units (RLU). The average RLU
measurement for each sample was expressed as a ‘relative luminescence’
(RLU/OD750). All samples were spaced one well apart in
the microplate to prevent any bleed through of luminescence signal from
one well to another.
Fluorescence analysis of mVenCP expression was performed using
200 μL of normalized cells. For each set of
measurements, samples were loaded into a black microplate in
triplicate along with a blank solution containing 200 μL of TAP medium.
Fluorescence was detected with the appropriate filter set formVenus (505 nm excitation, 540 nm emission) and expressed as
relative fluorescence units (RFU). The averaged blank RFU measurement
was subtracted from all samples to give the final value expressed as
‘relative fluorescence’ (RFU/OD750).