Mda lysis buffer

The assays regarding the detection of Malondialdehyde (MDA) and Superoxide Dismutase (SOD) activity were performed to analyze oxidative stress. For lipid peroxidation MDA assay, about 2 × 10⁶ HPAEpiCs were harvested after LPS treatment and/or cell transfection. The cells were washed using PBS and lysed using MDA lysis buffer (Abcam, Cambridge, MA, USA). Cell supernatant was collected by centrifugation, and incubated with thiobarbituric acid (Abcam) at 95 °C for 1 h, followed by analysis using a microplate reader (Thermo Fisher). For SOD activity assay, HPAEpiCs were collected and lysed using lysis buffer (Abcam), followed by centrifugation to harvest cell supernatant. The following assay was performed inferring to the instruction of a SOD activity assay kit (Abcam). The output of samples was determined on a microplate reader (Thermo Fisher).

Oxidative stress (malondialdehyde [MDA]) levels were measured in RTECs and kidney tissues using an MDA assay kit (Abcam, Cambridge, MA, USA). 10 mg of RTECs were homogenized on ice in 300 μl of MDA lysis buffer (Abcam, Cambridge, MA, USA), then centrifuged (13,000 × g, 10 min) to remove insoluble materials. 10 ml of plasma were mixed with 500 μl of 42 mM H₂SO₄ and 125 μl of phosphotungstic acid solution at RT for 5 min. After centrifuging (13,000 × g, 3 min), the pellet was resuspended on ice with 100 μl of double-distilled H₂O. Then, 200 μl of solution and 600 μl of 2-thiobarbituric acid solution were incubated at 95 °C for 60 min before cooling to RT in the ice bath for 10 min. The intensity of absorbance at 532 nm was proportional to the MDA level.

Lipid peroxidation (malondialdehyde (MDA)) levels in rats’ testis tissue and plasma were detected by MDA Assay Kit (Abcam). Ten milligrams of tissue were homogenized on ice in 300 μl MDA Lysis Buffer (Abcam) and then centrifuged (13,000 × g, 10 minutes) to remove insoluble materials. Ten microliters of plasma were mixed with 500 μl of 42 mM H₂SO₄ and 125 μl phosphotungstic acid solution at room temperature for 5 minutes. After centrifuging (13,000 × g, 3 minutes), the pellet was resuspended on ice with 100 μl double-distilled H₂O. Then 200 μl solution and 600 μl 2-Thiobarbituric acid solution were incubated at 95°C for 60 minutes, before cooling to room temperature in the ice bath for 10 minutes. The intensity of absorbance at 532 nm was proportioned to the MDA level.