Oxiselecttmin vitro ros rns assay kit

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)].

ROS are molecules that contain oxygen and that easily reacts with other molecules in a cell, and they induce deleterious effects to cells. ROS concentration was determined by an OxiselectTM in vitro ROS/RNS Assay kit (Cell Biolabs, San Diego, CA, USA). The assay was performed according to the manufacturer’s instructions. Briefly, the media from each group were collected to measure the free radical presence in the samples. All samples were transferred into 1.5 mL tubes and centrifuged at 10,000× g for 5 min. Each sample was added to wells of the designated plate. Then, 50 μL of the catalyst was added to each well followed by incubation for 5 min at room temperature. Lastly, 100 μL of DCFH solution was added to each well, and incubation was performed for 15 min at room temperature. The fluorescence intensity was measured using a fluorescence plate reader at 480 nm excitation/530 nm emission (Sunrise, Tecan, Hayward, CA, USA).

Total free radical activity was measured using the Oxiselect^TM In Vitro ROS/RNS assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacture’s protocol. Briefly, DCF standards were prepared in a concentration range of 0–10 μM by diluting the 1 mM DCF stock in 1 × PBS. Then, 50 µL of tissue lysates or DCF standard was added into a 96-well black-bottomed fluorescence plate (Nunclon^TM, Thermo Fisher Scientific, Roskilde, Denmark), and 50 µL of 1 × catalyst was added into the wells. Then the wells were mixed and incubated for 5 min at room temperature. DCFH solution (100 µL) was added to the wells and incubated at room temperature for 45 min in the dark. The fluorescence was measured using a GloMax^® Explorer (Promega, Madison, WI, USA) at 480 nm excitation and 530 nm emission. This assay employs a proprietary quenched fluorogenic probe, dichlorodihydrofluorescin DiOxyQ (DCFH-DiOxyQ), which is a specific ROS/RNS probe. The probe reacts with ROS/RNS species, which are rapidly oxidized to fluorescent DCF. Fluorescence intensity is proportional to the total ROS/RNS levels within the sample.