Transient transfection
HEK293T cells were seeded into a dish (10 cm, i.d.) at 106 cells, and were incubated overnight. Plasmids of pcDNA3-N-Flag-NLRP3 or pEGFP-C2-NLRP3 obtained from Addgene were transfected using TurboFect transfection reagents (Thermo Fisher Scientific) for 24 h. The transfected cells were seeded into a dish (5 cm, i.d.) at a density of 5 × 105 cells, and were incubated overnight. The cells were pretreated with YDZG (20 μM) for 1 h and stimulated with LPS (1 μg·mL-1) for 6 h, then treated with ATP for 30 min before harvesting.
MTT assayRAW264.7, J774A.1, and THP-1 cells were seeded into 96-well plates at a density of 4× 104, 1× 104, 2× 104 cells per well and were incubated overnight. Then, the cells were treated with different concentration of YDZG for 24 h, and an MTT assay was used to determine cytotoxicity according to our previous report(Gao et al., 2015).
Determination of NORAW264.7 cells were plated in 24-well plates at a density of 5 × 105 per well overnight. The cells were pretreated with YDZG (20, 40 or 80 μM) for 1 h, then LPS (1 μg·mL-1) was added to the YDZG containing medium, followed by 18 h culture. Treated cells were collected and stained with DAF-FM (1 μM) for an additional 1 h. The fluorescence signal was determined by FACScan flow cytometry using the FITC or PE channel.