Cy5651

The antibodies included TLR4 (1:100, GB11519, Servicebio, Wuhan, China) and NLRP3 (1:100, CY5651, Abways Technology, Shanghai, China). The images were taken by Sideview VS200 (Olympus, Japan) and were quantified with Image J.

EPCs (5 × 10⁴ cells) were seeded on sterilized coverslips. After treatment, cells were washed three times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO4, 1.8 mM KH₂PO4) and fixed by 4% paraformaldehyde (PFA, C104188, ALADDIN, Shanghai, China) for 30 min. The cell membrane was perforated by 0.3% Triton X-100 (Biosharp, BS084, Hefei, China) for 15 min, and the cell was blocked with 5% bovine serum albumin (BSA, Solarbio, A8010, Beijing, China) for 1.5 h. The primary antibodies rabbit anti-NLRP3 (1:200, Abways Technology, Inc., CY5651, Shanghai, China), rabbit anti-PYCARD (ASC, 1:100, Abways Technology, Inc., AY0406, Shanghai, China), rabbit anti-CD31 (1:100, affinity, P16284, Changzhou, China), rabbit anti-α-tubulin (1:1000, Abways, AB0048, Shanghai, China), rabbit anti-γ-H2AX (1:100, Proteintech, 10856-1-AP, Wuhan, China) were incubated for 5 h, respectively. After triple washed by PBS, NLRP3, and CD31 were labeled by the second fluorescent antibody Cy3–conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (1:100, Proteintech, SA00009-2, Wuhan, China), and ASC, α-tubulin, γ-H2AX were labeled by the second fluorescent antibody Goat Anti-Rabbit IgG (H + L) Alexa Fluor 488 (1:100, Abways Technology, Inc., AB0141, Shanghai, China) for 2 h, respectively.