Fixation permeablization buffer

Single-cell suspensions were washed twice with ice-cold flow cytometry staining (FACS) buffer (2% FBS + 1 mM EDTA in PBS), incubated with blocking buffer (1:100, 564765, BD Bioscience, Franklin Lakes, NJ, USA) for 15 min at 4°C. For cell surface antigen staining, single-cell suspensions were stained with CD45 (1:100, 368511, BioLegend, San Diego, CA, USA) and CD11b (1:100, 101228, BioLegend) in the dark for 1 h on ice and then washed two to three times with FACS buffer. For intracellular cytokine detection, cells were fixed and permeabilized with fixation/permeablization buffer (eBioscience, Waltham, MA, USA) according to the manufacturer’s protocol. Cells were then stained with NLRP3 (1:100, IC7578S-100UG, Novus, St. Louis, MO, USA), C1QA (1:100, NB100-64597, Novus), and PRDM1 (1:100, 565276, BD Bioscience) antibodies. Data were acquired within 2 h after staining on an LSR Fortessa (BD Biosciences), and analysis was performed by using FlowJo software (Tree Star, Ashland, OR, USA). Calculations made in bar graphs were generated in GraphPad Prism.

Cultured cells or cells harvested from peripheral blood were stained with fluorochrome-conjugated anti-rat CD4, CD25, CD80, CD86, CD11c, CD11b, IL-4, MHC-II, IFN-γ, and ROR-γt, Foxp3 Abs (BD Biosciences) and analyzed for expression of various cell surface or intracellular markers using a FACSCalibur platform (BD Biosciences). For intracellular cytokine (IL-4, IFN-γ) detection, cells were cultured with cell stimulation cocktail (protease inhibitor) for 16 h, then fixed and permeabilized with Fixation/Permeablization Buffer (eBioscience, MA) according to the manufacturer’s protocol. CD4⁺ T cell subsets were defined by expression of CD4 and IFN-γ for T helper (Th)1, expression of CD4 and IL-4 for Th2, expression of CD4 and ROR-γt for Th17 and expression of CD4, CD25, and Foxp3 for Treg. Cell apoptosis was tested using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences). For the enumeration of peripheral blood DC and CD4⁺ T cell subsets, CountBright absolute count beads (ThermoFisher Scientific, MA) were mixed with the cell samples and assayed via flow cytometry. Data were analyzed using FlowJo software (Tree Star, OR).