Fluostar optima

As described previously,^{23 (link)} Evans Blue (600 µg/animal), DyL-LDL, or DyL-HDL (each 500 µg/animal) were injected in the tail vein of S1P₃-iECKI, and CTRL mice 15 min prior to the i.p. injection of LPS (25 µg/animal). Mice were sacrificed after 3 h, and their peritoneal cavities were washed with 10 mL of ice-cold heparinized PBS. The cells were spun down, and the supernatants were analysed for Evans Blue with photometry (620 nm, FluoStar Optima, BMG LabTech, Ortenberg, Germany) and for DyL-LDL or DyL-HDL with fluorescence spectrometry (560 nm/590 nm, FluoStar Optima).

The level of superoxide (O²⁻) was measured at the end of the experimental protocol by dihydroethidium (DHE) staining, which can visualize the amount of superoxide present in the cells. DHE exhibits blue fluorescence in the cytoplasm and upon oxidation, it intercalates into the DNA and switches to a bright red fluorescence. The fluorescent intensity of each well was detected by a fluorescent plate reader (FluoStar Optima, BMG Labtech) in a well scanning mode (scan matrix: 10 × 10; scan diameter: 10 mm; bottom optic; no of flashes/scan point: 3; temp: 37 °C; excitation wavelength: 530 nm; emission wavelength: 620 nm).
The total reactive oxygen species (ROS) content was measured by cell-permeant 2′-7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), which is a chemically reduced form of fluorescein and used as an indicator of ROS level in cells. Upon cleavage of the acetate groups by intracellular esterases and oxidation, the non-fluorescent H₂DCFDA is converted to the highly fluorescent 2′-7′-dichlorofluorescein (DCF). The fluorescent intensity of each well was detected later by a fluorescent plate reader (FluoStar Optima, BMG Labtech) in a well scanning mode (scan matrix: 10 × 10; scan diameter: 10 mm; bottom optic; no of flashes/scan point: 3; temp: 37 °C; excitation wavelength: 480 nm; emission wavelength: 520 nm).

Antioxidant activity of essential oils was determined with oxygen radical absorbance capacity (ORAC) methods, as described by Dávalos et al. [95 (link)]. Previously, the essential oils were diluted with ethanol (1%). The assay was carried out in 75 mM phosphate buffer (pH 7.4) with the final reaction volume of 200 μL, including 20 μL of the diluted sample, blank or Trolox (used as standard curve 1–8 μM Trolox in each assay), 120 μL of fluorescein solution and 60 μL of AAPH solution, dispensed in a 96-well microplate (NUNC A/S, Roskilde, Denmark) and loaded into a microplate reader (FLUOstar Optima, BMG, Labtech Inc., Durham, NC, USA). The fluorescence was recorded every minute for 104 min (kex = 485 nm, kem = 520 nm). ORAC values were expressed as μmol Trolox equivalents/g of essential oil and were calculated from the regression equation of Trolox concentrations and net area under the fluorescence decay curve, which was generated by FLUOstar Optima software (V2.1 0 R4, BMG Labtech Inc., Durham, NC, USA). All samples were analyzed in triplicate.

By using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method, the radical scavenging activity of the methanolic OP1, OP2 and OP3 extracts was measured [67 (link)]. In a 96-multiwell plate, 50 μL aliquot of each OP extract (0–2 mg/mL) or of the standard Trolox (0–100 mg/mL), in triplicate, was added to 200 μL of DPPH solution (0.1 mM in methanol). After incubation in darkness for 30 min at 37 °C, the absorbance was measured at 490 nm using a UV–VIS microplate reader (FLUOstar Optima, BMG Labtech, Ortenberg, Germany) against DPPH solution as a blank. Values were expressed as Trolox equivalent (μg TE/mg dry extract).
The radical cation scavenging activity of each extract was measured using the 2-2′- azino-bis (3-ethylbenzo-thiazoline-6-sulphonate) diammonium salt (ABTS) method [68 (link)]. In a 96-multiwell plate, 50 μL aliquot of sample (0–5 mg/mL) was added to 200 μL of ABTS solution (5 mM). ATBS solution was derived by oxidizing ABTS with MnO₂ in distilled water for 30 min in the dark, and then the solution was filtered through filter paper. After 20 min incubation in darkness at room temperature, the absorbance was read at 734 nm using a UV–VIS microplate reader (FLUOstar Optima, BMG Labtech, Ortenberg, Germany) against ABTS solution as a blank. Values were expressed as Trolox equivalent (μg TE/mg dry extract).

Algal cell density and optical density at 730 nm were measured using a Z2 particle count analyser (Beckman Coulter) with limits of 2.974–9.001 μm, and a FluoStar Optima (BMG labtech) or Thermo Spectronic UV1 spectrophotometer (ThermoFisher), respectively. Colony‐forming units (CFU) of algal cells was determined by 10‐fold serial dilution of a culture aliquot in growth media and spotting 10 μl volumes on TAP + 1000 ng·L⁻¹ B₁₂ 1.5% agar plates, followed by incubation at 25°C, 20 μmol·m⁻²·s⁻¹ of continuous light for 4 days and then counting the number of colonies at 100× magnification. M. loti and E. coli CFU densities were measured similarly but on TY or LB plates with incubation at 28°C in the dark for 3 days or 37°C overnight, respectively. E. coli ED662 (ΔbtuF) was distinguished from ED656 by its kanamycin resistance and hence its ability to grow on plates containing 50 μg·ml⁻¹ kanamycin. Single cells for all bacteria were too small to count with a light microscope. When bacteria were grown axenically, optical density was also used as a proxy for cell density with measurements at 600 nm on a FluoStar Optima (BMG labtech) spectrophotometer.