Spectra max 180

ROS/RNS assay was determined using a fluorescence kit (STA-347, OxiSelect^TM in vitro ROS/RNS assay kit, Cell Biolabs, Inc., San Diego, CA, USA) at excitation and emission wavelengths of 480 and 530 nm, respectively, with a SpectraMax Gemini XS Fluorimeter. To assess lipid peroxidation in SD rat testes, MDA levels in testis tissue homogenates were measured using a commercially available kit (NWLSSTM Malondialdehyde Assay kit; Northwest Life Science Specialties LLC., Vancouver, WA, USA) following the manufacturer’s instructions. MDA forms a pink complex in aerobic conditions after incubation with thiobarbituric acid (TBA) at 60 °C. Absorbance of the colored complex was measured by kinetic spectrophotometric analysis at 532 nm using a Spectra Max 180 (Molecular Devices, Sunnyvale, CA, USA). MDA concentration in the sample was analyzed by comparing the measured absorbance value to an MDA standard curve [14 (link)]. MDA concentrations were normalized to total protein content [15 (link)].

Malondialdehyde (MDA) levels in testis tissue homogenates were determined using a commercially available MDA assay kit (NWLSSTM malondialdehyde assay kit; Northwest Life Science Specialties LLC., Vancouver, WA, USA) according to the manufacturer’s instructions. Absorbance of colored complex was measured at a wavelength of 532 nm by kinetic spectrophotometric analysis using a Spectra Max 180 (Molecular Devices, Sunnyvale, CA, USA). MDA concentration in the sample was analyzed by comparing the measured absorbance value to the standard curve of MDA. The level of MDA was expressed as μmole per mg tissue. ROS/RNS levels in testis tissue homogenates were determined using a fluorescence kit (STA-347, OxiSelect^TM in vitro ROS/RNS assay kit, Cell Biolabs, Inc., San Diego, CA, USA). Absorbance values were measured at excitation and emission wavelengths of 480 and 530 nm, respectively, with a SpectraMax Gemini XS Fluorimeter, as described previously [26 (link)].

Testis tissue malondialdehyde (MDA) levels were measured using an MDA assay kit as per the manufacturer’s instructions (NWLSSTM Malondialdehyde Assay kit; Northwest Life Science Specialties LLC, Vancouver, WA, USA). Briefly, MDA reacts with thiobarbituric acid forming a colored complex which was measured at an absorbance of 532 nm by a Spectra Max 180 (Molecular Devices, Sunnyvale, CA, USA). The MDA level is expressed as μmole MDA per mg of wet tissue. Testis tissue ROS/RNS levels were detected using an OxiSelect in Vitro ROS/RNS Assay Kit per the manufacturer’s instructions (STA-347, Cell Biolabs, Inc., San Diego, CA, USA). Briefly, absorbance was measured at 480 nm excitation/530 nm emission with a SpectraMax Gemini XS Fluorimeter (Molecular Devices, Sunnyvale, CA, USA) as described previously [20 (link)]. The activities of superoxide dismutase (SOD) (706002, Cayman Chemical, Ann Arbor, MI, USA), glutathione peroxidase (GPx) (703102, Cayman Chemical, Ann Arbor, MI, USA), and catalase (707002, Cayman Chemical, Ann Arbor, MI, USA) in whole testis tissue supernatants were determined using commercially available kits per the manufacturer’s instructions. SOD, GPx, and catalase activities are expressed per milligram of protein.