Powerwave xs2 microplate spectrophotometer

Seedlings were continuously grown for 14 days on media adjusted to various pH values. Five shoot samples were collected, weighed, and immediately placed in an Eppendorf tube with steel beads, immersed in liquid nitrogen and stored at −80 °C until analyzed. For analyzing chlorophyll concentration, samples were homogenized with a TissueLyzer II (Qiagen). Chlorophyll was extracted and measured following a protocol from Mackinney (19 ). Frozen tissues were dissolved in 500 μl of 80% (v/v) acetone. The tubes were centrifuged at 13,200 rpm for 5 min, and the supernatant was collected in the fresh amber Eppendorf tube. The 80% acetone step was repeated thrice or until the pellet became white, and all extracts were pooled together. Finally, the pooled extract was mixed thoroughly and centrifuged again to pellet down the remaining cell debris. The absorbance of 200 μL extract was measured at 663, 647, and 750 nm in a PowerWave XS2 microplate spectrophotometer from BioTek. Absorbance at 750 nm was used to correct the absorbance at 663 and 647 nm. Chlorophyll concentration was calculated using the equation described by Lichtenthaler (20 ).

Protein extraction from transduced cells was performed using Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA)^{49 (link)}. Samples were sonicated for 15 s at 10 mA, followed by centrifugation at 16,000 RCF for 10 min at 4 °C, and transferal of supernatant to a new tube. Samples were quantified with the DC Protein Assay (Bio-Rad, Hercules, CA) using the recommended protocol. Sample absorbance at 750 nm was quantified using the PowerWave XS2 Microplate Spectrophotometer (BioTek, Winooski, VT).

Cells were seeded in 96-well plates at a density of 2×10⁴ cells/ml with 100 μL of medium per well and then incubated with various concentrations of radotinib (0, 1, 10, and 100 μM) for 72 h at 37°C. The CellTiter 96 solution (20 μL) was added directly to each well and plates were incubated for 4 h in a humidified 5% CO₂ atmosphere at 37°C. Absorbance was measured with a PowerWave XS2 Microplate Spectrophotometer (BioTek, Winooski, VT) at 490 nm and the results were expressed as percentage changes from the basal condition using four to five culture wells for each experimental treatment. In some experiments, HL60 cells were cultured with 100 nM ATRA and 1 μM dasatinib for 4 days, and 10 μM radotinib was added to each group according to the planned schedule.

Lipid peroxidation was assessed in circulation and tissues. Plasma oxidized LDL concentration was measured using the corresponding ELISA kit (MyBioSource Inc., San Diego, CA, USA) in a PowerWave XS2 microplate spectrophotometer (Biotek Instruments Inc., Winooski, VT, USA). Conjugated diene hydroperoxide amount in perigonadal adipose tissue and the liver was measured as previously described [79 (link)] using a Beckman DU-640 UV-Vis spectrophotometer (Beckman Instruments Inc., Palo Alto, CA, USA). TBARS amount in plasma, erythrocytes, perigonadal adipose tissue and the liver was measured according to the method developed by Buege and Aust [80 (link)] with modifications described by Richard et al. [81 (link)] using an LS55 fluorescence spectrophotometer (Perkin Elmer, Shelton, CT, USA).
Protein carbonylation in plasma and the liver was measured as previously described [79 (link)] by labeling carbonyl-modified proteins with fluorescein-5-thiosemicarbazide (FTSC) and resolving and quantified them using a one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A culture medium for L. sakei (SAK-medium) was initially used, having the following composition: meat extract, ME (VWR) 8 g/L; yeast extract, YE (Costantino srl, Turin, Italy) 8 g/L; arginine, ARG (Merck K GaA) 0.5 g/L; Tween-80 (Merck K GaA) 0.5 mL/L, minerals and vitamins mix 1 mL/L as reported by Mapelli et al. [27 (link)]. Ingredients were dissolved in liquid CWP, kindly supplied by Latteria Soresina (Soresina, Italy).
Freshly produced CWP was collected at the cheese factory from the UF plant that processes whey from hard cheese manufacture, immediately frozen and kept at −20 °C until use. The CWP batch had a lactose content of 42 g/L and pH 6.2 and was filter sterilized just before use.
Both L. sakei growth and sakacin A production were determined in the SAK-medium and in MRS, being the latter taken as benchmark. L. sakei growth was studied at 26°C with different percentage of inoculum, i.e., 0.1, 0.5, 1 and 5% (v/v). Culture turbidity (OD) was measured spectrophotometrically at 600 nm every 15 min in a PowerWave™ XS2 Microplate Spectrophotometer (BioTek, Winooski, VT, USA); lag phase (min) and maximum growth rate (OD/h) were determined fitting data through the Baranyi and Roberts model [31 (link)] on DMFit 3.5 Excel add-in.

The absorbance spectra of melatonin, 5-MCA-NAT, IIK7, and agomelatine (1 mM) were determined at 25 °C using a 96-well quartz microplate (Hellma Analytics GmbH, Müllheim, Germany) and a Power Wave XS2 Microplate Spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA) between 200 and 500 nm. None of them presented an absorption above 370 nm. The spectra had an absorption maximum for melatonin and 5-MCA-NAT corresponding to the 230 nm wavelength. Likewise, the maximum absorbance for IIK7 and agomelatine were at 220 nm and 275 nm, respectively. Thus, these wavelengths (230 nm, 220 nm, and 275 nm) were used to detect melatonin and its analogs in drug release and uptake studies.