Cytokine specific elisa

Supernatants were subjected to ELISA for secreted cytokines IL-17A, IFN-γ, and IL-2 and quantified by cytokine-specific ELISA (BD PharMingen) according to the manufacturer’s guidelines. Serum cytokine concentrations were determined using BioLegend’s LEGENDplex bead-based immunoassays according to the manufacturer’s protocol 6 d after allo-HCT. Data were acquired on a FACS Canto II (BD Biosciences) flow cytometer and analyzed with LEGENDplex Data Analysis software.

To assay the induction of inflammasomes in lymph nodes, aPNMs (50 µg) and carboxyl-terminated γ-PGA nanomicelles were inoculated into the footpad of 6-week-old C57BL/6 mice and NLRP3 knockout mice after priming with LPS (5 µg) for 24 hours. Next, 3 and 6 hours after aPNM injection, the lymph nodes of the mice were removed. The dissected lymph nodes were transferred to round-bottomed microfuge tubes and snap-frozen in liquid nitrogen. Next, 300 µL of ice-cold lysis buffer (R0278; Sigma-Aldrich) was added, and the lymph nodes were homogenized by using an electric homogenizer (Z359971; Sigma-Aldrich). Another 600 µL of lysis buffer was added during homogenization. After homogenization was completed, the contents were stirred for 2 hours at 4°C. The supernatants were collected following centrifugation at 16,000× g for 20 minutes at 4°C. IL-1β was analyzed by using cytokine-specific ELISA (BD Biosciences) according to the manufacturer’s instructions.