Normal goat serum (ngs)

CFs cultured alone or in the presence of CMs were cultured on glass coverslips in 6-well plates. The cells were subjected to H/R as described above. Then, the cells were incubated with 4% fixative solution (Solarbio, China) for 30 min and blocked in 10% normal goat serum (Solarbio, China)/PBS for 30 min. The cells were incubated overnight at 4 °C with anti-NLRP3 (1:200), anti-ASC (1:200), anti-cleaved-caspase1 (1:200), anti-sarcomeric alpha actinin (1:200, Abcam, USA) and anti-Vimentin (1:200, Abcam, USA) primary antibodies. Thereafter, the cells were washed with PBS and incubated with goat anti-rabbit IgG/FITC (1:500, MultiSciences, China), goat anti-chicken IgG/Cy5 (1:500, Solarbio, China), and Cy3-conjugated Affinipure goat anti-mouse IgG (1:50, Proteintech, USA) secondary antibodies (30 min, room temperature) to visualize NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase1, Vimentin and sarcomeric alpha actinin. We used 4′,6-diamidino-2-phenylindole (DAPI) (1:200, Beyotime, China) to visualize the nuclei. Finally, the fluorescence was visualized using a fluorescence microscope. Three fields of view were randomly selected to observe the fluorescent protein in each batch of cells by one blinded for allocation.

For astrocytes, 2.4~2.5×10⁵ cells were seeded in a 24-well plate and grown to 50%-70% density. Then, cells were washed with PBS three times, treated with 4% paraformaldehyde (Solarbio) for 15 min, 0.5% Triton X-100 for 10 min, and then blocked with 10% normal goat serum (Solarbio) for 1 h. Next, the cells were incubated with primary antibody at 4 °C overnight. The next day, samples were incubated with secondary antibodies for 30 min at room temperature. Finally, Antifade Mounting Medium with DAPI (Beyotime Biotechnology) was used for sealing.
Spinal cord tissues were sectioned after paraffin embedding. After treatment with an environmentally friendly transparent dewaxing liquid (Solarbio) for deparaffinizing, slices were hydrated in 100%, 95%, 90%, 80%, and 70% ethanol solution in series. Next, sections were heated in citrate buffer for 15 min for antigen retrieval. The staining procedure was the same as that performed in cells.
An upright fluorescence microscope (Olympus, Tokyo, Japan) was used to obtain images.

A549 cells were seeded on coverslips and transiently transfected with the corresponding plasmids with Lipofectamine 2000 (Invitrogen, USA). Twenty-four hours later, the cells were washed in PBS, fixed in 4% formaldehyde for 15 min, and permeabilized with PBST (0.5% Triton X-100 in PBS) for 10 min at room temperature. The cells were washed three times with PBS and incubated in 10% normal goat serum (Solarbio, China) for 30 min. Primary antibodies (anti-CCR1, anti-CX3CR1) (catalog no. PA1-41062 [Thermo, USA] and 13885-1-AP [Proteintech, China]) were diluted with PBS containing 10% goat serum, and the cells were incubated with primary antibodies for 1 h at room temperature. After three washes, the cells were incubated with secondary antibodies containing Alexa 488-labeled goat anti-rabbit antibody (ab150080, 1:500; Abcam, UK). Coverslips were mounted on the glass slides with DAPI (4′,6-diamidino-2-phenylindole) Fluoromount-G (Southern Biotech, USA) for fluorescence microscopy or confocal imaging (Leica TCS SP5).