4 hne elisa kit

Blood samples collected from the experimental animals were centrifuged at 1000× g for 15 min to obtain serum. IL-1β, LPS, TNF-α in serum were measured by mouse IL-1β ELISA Kit (Neobioscience, Shenzhen, China), mouse LPS ELISA Kit (CUSABIO, Wuhan, China), and mouse TNF-α ELISA Kit (Neobioscience, Shenzhen, China), respectively.
bEnd.3 cells were treated with HSYA (10 μM) or NAC (2 mM) in the presence of LPS (100 ng/mL) for 16 h, referring to the dose in the published literature [11 (link),13 (link)]. The cell supernatant was collected and lactate concentration was measured according to the manufacturer’s instructions (Jiancheng, A019-2-1, Nanjing, China). Additionally, the treated bEnd.3 cells were washed with PBS and dissociated with trypsin, then the freeze–thaw process was repeated to lyse cells. After centrifuging at 1500× g for 10 min at 4 °C, the supernatant was collected for the measurement of 4-HNE according to the manufacturer’s protocol of the 4-HNE ELISA Kit (Elabscience, Wuhan, China).

The 4-HNE levels were measured using a 4-HNE ELISA kit (Elabscience Biotechnology, Bethesda, MD, USA), according to the manufacturer’s instructions. In brief, the cell pellets were washed and centrifuged for 10 min at 1500× g at 2–8 °C. After removal of the cell fragments, the supernatant was collected to carry out the assay. We added 50 μL standard or sample with 50 μL Biotinylated Detection antibody working solution to each well and incubated the, for 45 min at 37 °C. Then, we added 350 μL of wash buffer, soaked for 1 min, and repeated 3 times. Subsequently, 100 μL of HRP Conjugate working solution was added to each well, and the plate was incubated for 30 min at 37 °C. The process was repeated 5 times. After that, we added 90 μL of Substrate Reagent, covered the plate to block light, and incubated it for 15 mins at 37 °C. Finally, we added 50 μL of Stop Solution and read the plate at 450 nm using a microplate reader (Spectramax iD5 multi-mode microplate reader, Molecular Devices, Sunnyvale, CA, USA).