No elisa kit

The materials of H. diffusa was purchased from Bozhou Chinese Medicine Processing Plant (Bozhou, Anhui, China) and identified by Doctor Jing Wang (School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China).
RAW 264.7 murine macrophages were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China). SCA, which was only used as the reference compound in the isolation and identification process, was purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Dulbecco’s modification of Eagle’s medium (DMEM, No. 12430-054) and fetal bovine serum (FBS, No. 10099141) were purchased from Gibco (Thermo Scientific, Waltham, MA, USA). LPS obtained from Escherichia coli O111:B4, was purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). The antibodies of iNOS), COX-2, IκB-α, p38 and ERK1/2 were purchased from Proteintech Group, Inc. (Chicago, IL, USA). The antibodies of p-IκB-α, p-p38, p-ERK1/2, JNK and p-JNK were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). CCK-8 was purchased from Dojindo (Kumamoto, Japan). The ELISA kits of IL-6, IL-β1 and TNF-α were purchased from Neobioscience Technology Company (Shenzhen, China). NO ELISA kit was purchased from Beyotime Biotechnology (Shanghai, China), and PEG₂ was purchased from Enzo Life Sciences (New York, NY, USA). All other reagents were of analytical grade.

The cytotoxic activity of CME was detected using a previously described method,^{15 (link)} while intracellular ROS production was detected with a DCFH-DA using another previously reported method.^{16 (link)} Briefly, RAW 264.7 cells were pre-treated with chemicals for 24 h, and then stimulated with LPS (100 μg mL⁻¹). After incubation for 24 h, the cells were stained with 10 μM DCFH-DA at 37 °C in the dark. After 0.5 h, the cells were washed with phosphate-buffered saline (PBS) twice, and then placed in an Ascent FL fluorescence plate reader (37 °C). Emission at 535 nm was measured after excitation at 485 nm for 20 min. For NO concentration detection, cells were cultured using the same method before collecting cell supernatants. NO concentration was detected using a commercial NO ELISA kit (Beyotime, Shanghai, China) according to the manufacturer's instructions.