Live dead viability assay kit

CCK-8 kit (Beyotime, China)was used to test the cytotoxicity and proliferation of cells cultured with APO, KGN, and PAKM. The live/dead viability assay kit (Us everbright, China) was used to conduct Live/dead assays. Human ADSCs were incubated with fluorescein diacetate (5 μM)/propidium iodide (20 μM) at 37 °C in the dark for 5 min. The fluorescent image was taken using fluorescence microscope (Leica). Annexin V-FITC/PI Apoptosis Detection Kit (Solarbio, China) was used to measure the cell apoptosis, and then analyzed by the flow cytometer (BD Biosciences, USA).

After hNPPCs were pre-modified with PEG-PIB, the live/dead viability assay kit (Us Everbright, China) was used to conduct Live/dead assays of PEG-PIB pre-modified hNPPCs at different pH values (6.2, 6.4, 6.8, and 7.4). Meanwhile, IF was used to examine pyroptosis-associated ASC expression within the rhodamine packaged PEG-PIB hNPPCs at different pH values (6.2, 6.4, 6.8, and 7.4) after co-culture for 24 h.
Then WB was used to assess the possible pathways underlying the inhibitory effect of PEG-PIB on hNPPCs pyroptosis. Groups were set as follows: the normal control (NC) group did not undergo treatment with acid stimulation and PEG-PIB; for the degeneration control (DC) group, the medium was adjusted to 6.2, and the PEG-PIB group was treated with acidic stimulation and PEG-PIB. All groups underwent Western blot analysis to quantify the nucleoprotein expression of NLRP3, ASC, Caspase1, IL-1β, IκBα (1:1000, ab10286, Abcam) and p-IκBα (1:1000, 2859T, Cell Signaling Technology) to identify the functional pathways associated with PEG-PIB.