No assay kit

BV-2 microglial cells were seeded into 6-well plates. When cell confluency reached approximately 60%, cells were starved for 6 h and then pretreated with low (15 μM), middle (30 μM) and high (60 μM) concentrations of NADD for 1 h, followed by co-treatment with or without 1 μg/mL LPS for 24 h. After that, the cell supernatant was collected to detect the production of NO in each group using the NO assay kit (Beyotime) according to the manufacturer’s instructions.

NO production represented by nitrite concentration in the supernatants of cultured cells in 96-well plates was assayed with Griess reaction using a NO Assay Kit (Beyotime, Shanghai, China). The absorbance was measured at the wavelength of 540 nm. The ROS levels in cells cultured in 24-well plates were measured using an oxidation-sensitive fluorescent probe DCFH-DA Kit, and Rosup was included as a positive control (Beyotime, Shanghai, China). The DCF fluorescence intensity in cells was measured using a Lionheart FX Automated Microscope.

The NO levels in supernatants of Raw264.7 cells (WT and CYP26A1^−/−) were determined using NO assay kit (Beyotime, S0021) according to the manufacturers’ protocols. Briefly, cells were seeded in 96-well plates at a density of 2 × 10⁴ cells/well and polarized to M1 for 12 h, 24 h, 36 h, 48 h and 72 h. Subsequently, 50 μL of supernatants were collected and mixed with equal volumes of Griess reagent I and II in a new 96-well plate. The NO concentrations were determined at 540 nm using automatic microplate reader (Bio Tek, PowerWave XS).

The concentrations of NO and ET-1 in the cell supernatant were detected using an NO assay kit (Beyotime Biotechnology, Shanghai, China) and Human ET-1 ELISA kit (Cloud-Clone Technology, Wuhan, China), respectively. The detection method of SOD and MDA content in the cells was shown as above.

The intracellular ROS levels of cultured cells in 24-well plates were assessed by the use of an oxidation-sensitive fluorescent probe DCFH-DA (Eruslanov and Kusmartsev, 2010 (link)), and Rosup was included as a positive control (Beyotime, Shanghai, China). The intensity of DCF fluorescence was assessed by the use of a Lionheart FX Automated Microscope. NO production represented by nitrite concentration in the supernatants of cultured cells was assayed with Griess reaction in 96-well plates (Schulz et al., 1999 (link)) by the use of a NO Assay Kit (Beyotime, Shanghai, China). The absorbance was measured at 540 nm wavelength.

Intracellular NO content was measured using the NO assay kit (Beyotime, Shanghai, China). First, the mycelium was frozen in liquid nitrogen and ground. Then, 20 mg was placed into a centrifuge tube, and 200 µL of lysate was added. After incubation in an ice bath for 10 min and centrifugation at 14,000× g for 10 min, the supernatant was collected. The NO concentration was detected according to the instructions of the kit.

PHI was prepared by our laboratory (purity > 98.0%). DSS (molecular weight of 36,000–50,000 Da) was obtained from MP Biochemicals (Santa Ana, CA, USA). The enzyme-linked immunosorbent assay (ELISA) kits (such as TNF-α, IL-1β, IL-6, and IL-10) and TUNEL assay kit were purchased from Boster Biological Technology (Wuhan, China). The activity assay kits of MPO, SOD, and MDA were purchased from Jiancheng Bioengineering Institute (Nanjing, China). The NO assay kit purchased from Beyotime Biotechnology (Shanghai, China). The antibodies such as p-Src (ab185617), p-p38 (ab195049), JNK (ab179461), p-JNK (ab124956), ERK1/2 (ab184699), p-ERK1/2 (ab201015), p65 (ab32536), p-p65 (ab76302), IκBα (ab76429), p-IκBα (ab133462), occludin (ab216327), E-cadherin (ab231303), and ZO-1 (ab190085) were purchased from Abcam (Cambridge, UK). The other antibodies of iNOS (A3200), TLR4 (A5258), and Src (A0324) were purchased from ABclonal Technology (Wuhan, China). The antibody p38 (D155224) and the MightyScript Plus First Strand cDNA Synthesis Master Mix were obtained from Sangon Biotechnology (Wuhan, China). SYBR green mix was purchased from Mei5 Biotechnology (Beijing, China). Triol reagent was obtained from Takara (Shiga, Japan).