H2dcfda probe

The production of ROS in neutrophils of different treatment groups were analyzed using the dichlorodihydrofluorescein diacetate (H2DCFDA) probe (S9687, Selleck, Houston, Texas, USA) by flow cytometry. Neutrophils (Neu) were stimulated with Fn, PMA, Fn + TAK-242, Fn + ML130, or Fn + GSK717 for 6 h, then cells were collected and washed with PBS and then stained with 10 µM H2DCFDA for 30 min at 37 °C. Finally, after washing off residual H2DCFDA, cells were analyzed using flow cytometry (CytoFLEX). To investigate apoptosis of CRC cells, which had been treated with (Neu)-CM, (Neu + Fn)-CM, (Neu + Fn + DNase I)-CM, (Neu + PMA)-CM or NETs for 48 h, Dead Cell Apoptosis Kit (Life Technologies) with Annexin V / FITC and PI was used according to the manufacturer’s recommendation. All the experiments were performed three times.

The production of ROS in THP-1 macrophages was measured using the dichlorodihydrofluorescein diacetate (H2DCFDA) probe (S9687, Selleck, USA) according to the manufacturer’s recommendation. In brief, THP-1 macrophages were stimulated with rhS100A8 (5 µg/mL) or the control protein GST (5 µg/mL) for 6 h. THP-1 macrophages were collected and washed three times with serum-free medium, and subsequently incubated in serum-free medium containing 10 µM H2DCFDA probe at 37 °C for 30 min in the dark. Then, the media containing H2DCFDA was removed and washed two times with PBS, and the fluorescence intensity of the cells was analyzed by flow cytometry (CytoFLEX). Cells that had been incubated without H2DCFDA were used as negative controls. To detect pyroptotic death in THP-1 macrophages that had been treated with rhS100A8 or control protein GST for 24 h, FLICA 660-YVAD-FMK (FLICA 660 in vitro Active Caspase-1 Detection Kit; ImmunoChemistry Technologies, Davis, CA, USA) was used according to the manufacturer’s instructions and propidium iodide (PI) was used to mark cells with membrane pores (Life Technologies, Carlsbad, CA, USA). Flow cytometry measurements were performed three times for each treatment. The mean fluorescence intensity was quantified usingFlowJo v10.8.1 (FlowJo LLC, Ashland, OR, USA).

The production of ROS in cells was measured using the H2DCFDA probe (S9687, Selleck, USA) according to the manufacturer’s recommendation. The stained cells were detected using a CytoFLEX cytometer instrument (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo v10.8 software. Dead and adherent cells were discriminated out by gating on live/dead population.