Propidium iodide staining

HUVEC-Cs were treated with PDA (10 μM), trypsinized and fixed with 70% ethanol. The cell cycle was analyzed using flow cytometry after propidium iodide staining (BD Biosciences).

Cobalt complex 2-treated cells were harvested and analysed by flow cytometry using propidium iodide staining (BD Biosciences, San Jose, CA, USA) according to the manufacturer instructions. Cell population in different phase of cell cycle were quantitatively counted by a flow cytometer (BD FACSAria III, San Jose, CA, USA). Data acquisition and analysis were performed with CellQuest (BD Biosciences, San Jose, CA, USA) from triple independent experiments.

Active Caspase-1 and PI fluorescence in vivo and in vitro are necessary for pyroptosis characterization. We measured the active caspase-1 level in cell suspensions with Caspase-1 Detection Kit (ImmunoChemistry Technologies, USA) using flow cytometry according to the manufacturer's instructions. And membrane pores induced by pyroptosis were marked by propidium iodide staining (BD, USA).

RAW264.7 cells were left alone or cultured with IL-4 (5 ng/ml) for 48 h. Some cells were also treated with 0.1 μM MC-LR or MC-RR. The cells were harvested, washed, and resuspended in 0.5% bovine serum albumin (BSA)-phosphate-buffered saline (PBS). Next, the cells were preincubated with mouse Fc block (eBioscience, San Diego, California, United States) for 15 min and subsequently stained with an Alexa Fluor 488-conjugated anti-CD11b Ab (eBioscience) and an allophycocyanin (APC)-conjugated anti-CD206 Ab (eBioscience) for 30 min at 4°C in the dark. Flow cytometric analyses were performed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, United States) and FlowJo software. Apoptotic cells were analyzed by flow cytometry using annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining (BD Biosciences). Some cells were fixed in 70% ethanol for overnight, stained with 10 μg/ml PI, and analyzed for cell cycle distribution by flow cytometry.

Propidium iodide (PI) staining (BD, Franklin Lakes, NJ) was used to quantify cell cycle analysis. SRA01/04 cells were fixed with 75% ethanol for overnight at -20° C before stained with PI. After washing with HBSS for three times, the stained cells were analyzed by a FACS Calibur Flow cytometer (BD, NJ, USA).