Dcfh da probe

According to the manufacturer’s protocol, DCFH-DA probes (Beyotime Biotechnology) were diluted in serum-free media to a final concentration of 10 μmol/L. The complete medium was replaced with the above probe-containing serum-free medium for 20 min at 37 °C, allowing the probes to enter the cells. Cells were collected then washed three times to remove the non-cell-derived probes. Then, the cells were resuspended with 200 μL of PBS and fluorescence intensity was detected by flow cytometry.

Cell apoptosis and ROS levels were evaluated using an Annexin V-FITC/PI Apoptosis Detection Kit (V13241, Invitrogen, USA) or DCFH-DA probes (S0033S, Beyotime, China) separately according to the manufacturer's instruction. Briefly, the HUVECs were seeded in 6 well at a density of 2 × 10⁵ cells/well and allowed to attach overnight. The next day, the medium was replaced with serum-free medium added 1.2 mM H₂O₂ for 24 h or not. Then, the cells were treated with drug. For cell apoptosis detecting, cells were coincubated with Annexin V-FITC and then propidium iodide for 15 min at 22°C and then detected by flow cytometry. For ROS detecting, cells were coincubated with DCFH-DA probes (10 μmol/L) at 37°C for 30 min and collected by centrifugation. After resuspended with PBS, DCFH-DA-labeled cells were further detected by flow cytometry.

Cells were prepared as previously described, and stained for 30 min at 4 °C with relevant antibodies. Cells were acquired and analyzed on a Verse flow cytometer (BD Biosciences). All analyzed immune cells were acquired by gate CD45. Local neutrophils were identified as CD11b⁺Ly6G^hi, mast cells were identified as CD117⁺FcεRI⁺, macrophages infiltrating in the colon were identified as CD103⁻CD11c⁻CD11b⁺ and the two subpopulations of the colon dendritic cells were identified as CD103⁺CD11c⁺CD11b⁻ or CD103⁺CD11c⁺CD11b⁺.The level of ROS in active neutrophils was detected by 0.1 mM DCFH-DA probes (S0033, Beyotime, China) for 30 min at 4 °C per the manufacturer’s instructions.