Synergy 2 spectrophotometer

NKA activity in gill homogenates was determined using a temperature-regulated microplate method^{39 (link)}. Ten microliters of samples were run in duplicate in 96-well microplates at 25 °C and read at a wavelength of 340 nm for 10 min on a BioTek Synergy 2 spectrophotometer (BioTek, Winooski, VT). Protein concentration of the homogenate was determined using a BCA protein assay (Thermo Fisher Scientific, Rockford, IL).
Plasma chloride was analyzed by the silver titration method using a Buchler-Cotlove digital chloridometer (LABCONCO, Kansas City, MO, USA) and external standards.

The content of photosynthetic pigments was determined on the 1st, 14th day of drought, panicle ejection phase, and kernel milk maturity phase by a modified spectrophotometric method according to Lichtenthaler and Wellburn [70 (link)]. Leaf samples (5 mg) were homogenized in 80% ethanol. The homogenate was centrifuged at 1000 g for 5 min at 4 °C (Eppendorf Centrifuge 5702 R centrifuge, Germany). Then, the supernatant was stored at 4 °C in the dark until absorbance was measured. The absorbance value was measured using a Synergy 2 spectrophotometer, BioTek (Winooski, VT, USA), at wavelengths λ = 470 nm, 648 nm and 664 nm. The contents of chlorophyll a and b and carotenoids were calculated using the following formula:



where Chl_a = chlorophyll a, Chl_b = chlorophyll b, Car = carotenoids, A₆₆₄ = absorbance at 664 nm, A₆₄₈ = absorbance at 648 nm; A₄₇₀ = absorbance at 470 nm. The concentrations was expressed in micrograms of a given pigment in 1 mL of extract.

To assess tracer and nanobody leak, animals were perfused in the left ventricle with PBS. Brains (and other organs) were then collected, and their weight measured. Next, brains (and other organs) were incubated in formamide (Sigma-Aldrich, F7503) at 56 °C for 48 h. Dye fluorescence was then measured using a spectrophotometer at the appropriate emission and excitation wavelength. Dye extravasation from Unc5B^fl/fl, Unc5BiECko, WT treated with CTRL anti-Unc5B-1 and anti-Unc5B-2 were performed at the Yale Cardiovascular Research Center (New Haven, CT, USA) on a BioTek synergy2 spectrophotometer. Dye extravasation from WT treated with CTRL IgG2b and anti-Unc5B-3 were performed at the Paris Cardiovascular Research Center (Paris, France) on a Flexstation3 spectrophotometer. All results were normalized to the corresponding brain weight and reported to a standard made of known concentrations of dye diluted in formamide. Results are shown as ng of dye per mg of corresponding organ or tissue.

After 24 hours of mechanical strain, conditioned media were immediately collected from the wells and stored at −20°C. ELISA kits were purchased from RayBiotech for stem cell factor, hepatocyte growth factor, platelet-derived growth factor, and vascular endothelial growth factor A. The insulin-like growth factor 1 ELISA was purchased from Thermo Fisher Scientific. Experiments were performed according to the manufacturer's protocol. Briefly, diluted conditioned media or standard was added to the appropriate well and incubated for 2.5 hours at room temperature. Following washing, the appropriate biotin-conjugated antibody was added to each well and incubated for 1 hour at room temperature. Following washing, a horseradish peroxidase-conjugated streptavidin solution was added to each well and incubated for 45 minutes at room temperature. After additional washing, substrate was added to each well and incubated for 30 minutes at room temperature. Finally, stop solution was added to each well. Plates were read immediately at 450 nm on a BioTek Synergy2 spectrophotometer.

To assess the oxidizing activity, we used the marker CM-H₂DCFDA (Life Technologies, Eugene, OR, USA), following the manufacturer’s recommendations. DU145 and PC-3 cells (1 × 10⁴/well) were seeded in black 96-well plates with a clear bottom, stabilized for 24 h, and treated with sulforaphane and/or vitamin D for 3 h. After exposing the cultures to CM-H₂DCFDA, H₂O₂ was added to the positive control wells for 20 min. Fluorescence was recorded in a Synergy 2 spectrophotometer (BioTek; Winooski, VT, USA) using excitation and emission filters set at 450 and 520 nm, respectively. The presented results show the percentage of reactive species in relation to the solvent control, with the fluorescence values of the solvent control being considered 100%.

The assay was performed as specified by the Autophagy Assay Kit’s manufacturer (ab139484; Abcam, Cambridge, UK). The HepG2 cells (1 × 10⁴ cells/well) were seeded in a 96-well plate, stabilized for 24 h, and treated for further 24 h. Fluorescence was recorded in a Synergy 2 spectrophotometer (BioTek^®; Winooski, VT, USA) with excitation/emission filters of 463/534 nm (green) and 350/461 nm (blue). The green fluorophore labels the autophagic vesicles that contain the LC3 protein while the blue fluorophore labels the cell nucleus. Fluorescence values are expressed as the autophagy ratio (ratio of green to blue fluorescence values) (Tusskorn et al., 2019). Images were acquired using the Leica DMI 6000B Fluorescence Microscope (Leica; Wetzlar, Hessen, Germany).

Intracellular and extracellular ATP levels were determined in triplicate cell cultures. Overnight cultures were washed in PBS and suspended in PBS with 10 mM glucose and either water (sham), 25 nM PlnEF or 25 µM nisin (Sigma). nisin was added at concentrations that limited growth of LP965 to a similar extent as 25 nM PlnEF. At indicated time points, 120 µl samples were collected and centrifuged for 2 min at 20,000X g. The supernatant (extracellular ATP) was aspirated and mixed at a ratio of 1:1 with dimethyl sulfoxide (DMSO, Sigma). The cell pellet (intracellular ATP) was then mixed with 100 µl DMSO. ATP concentrations were determined with the Invitrogen ATP Quantification kit according to manufacturer's instructions and luminescence was quantified according to a standard curve on a Biotek Synergy 2 spectrophotometer.