Blood samples were collected in lithium-heparin tubes and were centrifuged at 1,500 × g for 10 minutes. Plasma was separated and stored at -80 °C until use. Index of oxidative injury was determined by the levels of malondialdehyde (MDA) using an MDA assay kit (A003-1-2, Nanjing Jiancheng Bioengineering Institute, Nanjing), whereas the antioxidant capacity was indicated by the levels of superoxide dismutase (SOD) using a SOD assay kit (A001-3-2, Nanjing Jiancheng Bioengineering Institute, Nanjing). The readings were measured by spectrophotometry.
Mda assay kit a003 1 2
Manufactured by Nanjing Jiancheng
Sourced in United States, China
The MDA assay kit (A003-1-2) is a laboratory tool used for the quantitative determination of malondialdehyde (MDA) levels. MDA is a widely used biomarker for oxidative stress. The kit provides the necessary reagents and protocols to perform colorimetric or fluorometric MDA detection and quantification.
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4 protocols using mda assay kit a003 1 2
1
Plasma Oxidative Stress and Antioxidant Capacity
2
Oxidative Stress Biomarkers Analysis
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H2O2 was purchased from Sinopharm Chemical Reagent Co. Ltd, Shanghai, China (30%, 10011218). TMAO was purchased from the Sigma-Aldrich, St. Louis, MI, USA (95%, 317594-5G), T-SOD assay kit (A001-1-2) and MDA assay kit (A003-1-2) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Details of the used antibodies are shown as follows: MyoD1 (ab64159, Abcam, Cambridge, UK), CAT (21260-1-AP, Proteintech, Wuhan, China) and GAPDH (10494-1-AP, Proteintech, Wuhan, China).
3
Sperm malondialdehyde assessment
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Sperm malondialdehyde (MDA) content was measured with MDA assay kit (A003-1-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the post-thaw sperm were ultrasonicated (20 kHz, 300 W, operating at 50%, 3 min for 10 s on and 5 s off) on ice. The sample was mixed with the pre-prepared reaction buffer reagent, boiled for 40 min, then centrifuged for collecting supernatant after cooling. The absorbance was measured at 532 nm with a microplate reader (TECAN, Infinite M Nano, Männedorf, Switzerland).
4
Oxidative Stress Markers in Testis Samples
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After homogenizing in cold physiological saline solution, testis samples were centrifuged at 500 g for 15 min to obtain supernatants for the biochemical analysis of oxidative stress markers. As described in a previous study [22 (link)], glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) were analyzed using commercial kits, according to the procedure provided by the manufacturer (GPx assay kit, A005-1-2; SOD assay kit, A001-3-2; CAT assay kit, A007-1-1; Nanjing Jiancheng Biological Engineering Research Institute Co. Ltd., Nanjing, China). Malondialdehdye (MDA) levels were measured using a colorimetric method (MDA assay kit, A003-1-2, Nanjing Jiancheng Biological Engineering Research Institute Co. Ltd.). The protein concentration of all samples was determined using the Bradford method [23 (link)].
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