Cary eclipse fluorescent spectrophotometer

A range of 0 mol, 0.05 pmol, 0.001 nmol, 0.002 nmol, 0.005 nmol, 0.01 nmol, 0.02 nmol of Ubi amount were coated inside the wells of Corning Costar® 96-Well EIA/RIA Stripwell™ and blocked with 2% BSA for an hour before addition of 0.1 nmol conjugated Ubi scFv-LPETG₅-eGFP and allowed to bind for an hour before washing and re-consitute the volume inside the well with 1 × PBS. To measure the fluorescent intensity, Agilent Cary Eclipse Fluorescent spectrophotometer was used. Emission peak of eGFP was determined using excitation taken from 460 nm and emission from 490 nm to 600 nm.

A previously described fluorometric assay (Krahn et al. 2012 (link)), involving the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)], was employed to measure the degree of CM depolarization promoted by AG treatment of P. aeruginosa. Briefly, early logarithmic phase (optical density at 600 nm [OD_600 nm] = 0.3–0.5) L-broth subcultures of P. aeurginosa were treated with the AGs gentamicin (2 or 5 μg/mL final concentration) or tobramycin (0.5 or 1.25 μg/mL final concentration) or the aminocyclitol, spectinomycin (SPC) (1280 μg/mL). In some experiments P. aeruginosa was pretreated with chloramphenicol (128 μg/mL) for 15 min prior to the addition of gentamicin. Samples (5 mL) of the AG-treated and untreated control cultures were taken immediately and then hourly over 3 h and exposed to DiBAC₄(3) (Invitrogen, Burlington, Ontario, Canada) at 37°C for 5 min in the dark at a final concentration of 10 μg/mL. Bacteria were then pelleted and resuspended in phosphate-buffered saline (Nehme et al. 2004 (link)) to a final OD_600 nm of 0.1. Membrane depolarization-dependent fluorescence emitted by cells was then measured using a Varian (now Agilent, Mississauga, Ontario, Canada) Cary Eclipse fluorescent spectrophotometer with excitation and emission wavelengths of 490 and 518, respectively.

All the fluorescence measurements were carried out with Cary Eclipse Fluorescent Spectrophotometer, Agilent technologies using quartz cells of path-length 10 mm. Both excitation and emission band slits were fixed at 10 nm and the scan rate was selected at 1800 nm/min. The excitation wavelength was selected at 425 nm, while emission spectra were collected in the range of 450–600 nm for curcumin and the excitation wavelength for quercetin was 440 nm while the emission was collected in the range 500–600 nm.