Varian cary 50 spectrophotometer

Isotropic absorption spectra (UV/Vis) were measured on a Varian Cary 50 spectrophotometer (Agilent Technologies Inc., Santa Clara, USA); fluorescence spectra were measured on an LS 50B luminescence spectrometer (PerkinElmer, Rodgau, Germany). CD and LD were measured on a J‐810 spectropolarimeter (Jasco Corp., Tokyo, Japan), which could be equipped with a microvolume Couette flow LD cell (Dioptica Scientific Limited, Rugby, Warwickshire, UK) with a 0.5 mm optical path length for the LD measurement.26 The LD spectra were independent of the angular velocity of the rotating cell. Rotating velocities up to 3000 rpm, corresponding to shear forces of about 1200 s⁻¹, were used. CD measurements were carried out at 20 °C; all other spectroscopic measurements were performed at room temperature ((22±1) °C). Cuvettes for UV/Vis, fluorescence and CD spectroscopy were siliconised before measurements, according to the “Siliconization of Glassware” protocol by using a silicone solution in isopropanol (SERVA Electrophoresis GmbH, Heidelberg, Germany).

The reaction between the nitroalkenes and the thiols βME and GSH was followed through the change in UV absorption of the nitroalkene, at 350 nm for NATOH and NATxME or 260 nm for NATx0. The fastest reactions were monitored in a SX20 stopped-flow spectrometer (Applied Photophysics); intermediate reactions (t > 10 s) were followed in a Varian Cary50 spectrophotometer (Agilent) using a RX2000 rapid mixing stopped-flow unit (Applied Photophysics); and the slowest reactions (t > 600 s) were studied using a Varioskan Flash plate reader (Thermo). The reactions were performed in TMA20 buffer at the specified pH and 25 °C. The time courses were fitted to a single exponential function (equation 1) and the resulting rate constants (k_obs) were plotted vs the thiol concentration to obtain the rate constants of addition (k_f) and elimination (k_r). $$\mathit{Abs}=\mathit{Amp}\times \exp \left(-{\mathit{k}}_{\mathit{obs}}\mathit{t}\right)+\mathit{C}$$

The prepared membranes were thoroughly washed and cut to the desired size and loaded into an ultrafiltration test cell. The surface area of the membranes was 0.785 cm² and they were initially pressurized to 3 Ba. The pressurized membranes were used in subsequent ultrafiltration experiments. The water content of the membranes was obtained after soaking the membranes in water for 24 h. They were weighed after being wiped with filter paper. The wet membranes were placed in an oven at 75 °C for 48 h, cooled to room temperature in a desiccator and weighed again. Water content percentage retained by the membrane, WC, was calculated based on the wet and dry membrane weight data, w₁ and w₂, respectively.
Subsequently, the studied membranes were used in the treatment of 50 mL of aqueous solutions containing 3 mg·L⁻¹ DCF. Crossflow filtration was used, the feed solution being passed over the studied membrane using a vacuum of 20 mBar, recirculating the permeate through the membranes for 9 cycles. The studies were performed at the natural pH of the solutions (pH ≈ 6, T = 23 °C). The concentration of DCF in the feed solution and in permeate was determined by UV–VIS spectrophotometry at the wavelength λ = 275 nm, using a Varian Cary 50 spectrophotometer (Agilent, Santa Clara, CA, USA).

The pH-metric titrations
were recorded at 25 °C on a Varian Cary 50 spectrophotometer
(Agilent) over the spectral range 200–900 nm and on a J-815
CD spectropolarimeter (JASCO) covering the spectral range of 228–850
nm with a 1 cm-path quartz cuvette (Helma). NiCl₂ solution
was added to the samples to reach a 1:0.9 peptide-to-metal ratio.
Titrations were performed in water by adding small amounts of concentrated
NaOH solution. The pH stability was checked for each titration point
and adjusted with small amounts of concentrated NaOH or HCl solutions.
Dilutions were included in the calculations.

Intracellular TAG content was measured using the “Triglycerides liquid” kit (Sentinel, Milan, Italy), as previously described [57 (link),58 (link)]. The absorbance was recorded at 546 nm using a Varian Cary50 spectrophotometer (Agilent, Milan, Italy). For measurement of extracellular TAG content, the culture media were processed according to the same method. Values were normalized to protein content, and data are expressed as percent TAG content relative to controls [59 (link)]. For intracellular lipid staining, cells grown on coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Neutral lipids were stained by incubation with 1 μg/mL BODIPY 493/503 (Molecular Probes, Life Technologies, Monza, Italy) in PBS for 30 min [60 (link)]. After washing, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), 5 μg/mL (ProLong Gold medium with DAPI; Invitrogen, MA, USA). Mounted slides were examined at 10X magnification by Olympus IX53 light microscope (Olympus, Milano, Italy), equipped with the standard epifluorescence filter setup. Representative images were captured with a CCD UC30 camera (Olympus) and digital image acquisition software (CellSens Entry, Olympus).

The cell population of LAB was determined by measuring the optical density at 600 nm (OD_{600 nm}) by using a Varian Cary 50 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).

Absorbance spectra were determined using a Varian Cary 50 spectrophotometer (Agilent Technologies). For pH titrations, proteins in 20 mM Tris-HCl (pH 8.0) were diluted (~ 100-fold) as required into selected 0.1 M buffers. Absorbance spectra were recorded at 25°C 30 s after mixing. Sample pH was monitored using a micro-pH probe. Fluorescence spectra were determined using a Varian Eclipse fluorescence spectrophotometer (Agilent Technologies). All spectra were corrected and were determined under illumination conditions that did not induce photoswitching.
Φ_F values were determined for proteins in 20 mM Tris-HCl pH 8.0, 300 mM NaCl at 25°C using solutions of Rhodamine 6G (Φ_F, 0.95) in ethanol as a standard [13 (link),29 ].