Uric acid assay kit

Mouse J774A.1 macrophage cells were grown in DMEM supplemented with 10% FBS (v/v), 100 U/mL of penicillin and 100 μg/mL of streptomycin. MTT assay was used to determine J774A.1 cell viability after the cells (2500 cells/well) were cultured with AH-NPs or AH-MPs (aluminum content, 173 μg/well) for 72 h. Briefly, 20 μL of MTT reagent (5 mg/mL) was added to the wells and incubated at 37° C in the dark for 3–4 h. Two hundred microliters (200 μL) of dimethyl sulfoxide was added into each well and incubated for an additional 15 min to solubilize the MTT-formazan product. Absorbance was measured at 570 nm. A cell viability of greater than 90% was considered non-toxic. Triton X-100 (0.001%, v/v) was used as a positive control.
To determine uric acid production, J774A.1 cells were incubated with the AH-NPs or AH-MPs as mentioned above, and uric acid concentration in the cell culture medium was determined using a Sigma-Aldrich uric acid assay kit following the manufacturer’s instructions.

DAMPs were isolated from ex vivo killed cells. To generate doxorubicin-induced cell death, 1×10⁶ cells were incubated for 4 h with doxorubicin (10 mM) (Sigma) followed by washing 5 times with fresh culture media. Cells were incubated for 2 days in 1 ml of culture media. Culture supernatants were collected, centrifuged for 5 min at 1200 RPM and stored at −80°C until use. To generate sonication-induced cell death, 1×10⁶ cells in 1 ml of Dulbecco’s phosphate-buffered saline (DPBS) (Sigma) were sonicated for 1.5 min with Branson Sonifier 250 (Branson Ultrasonics, Danbury, CT). The levels of extracellular DNAs (exDNAs), HMGB1, adenosine triphosphate (ATP) and uric acid in the DAMPs were determined using Quant-iT PicoGreen DNA assay kit (ThermoFisher, Waltham, MA), HMGB1 ELISA kit (Tecan, Morrisville, NC), ATP determination kit (ThermoFisher) and Uric Acid Assay kit (Sigma), respectively, by following the manufacturer’s instructions. For circulating DAMPs in human blood, sera from citrated blood samples were collected from 3 patients with polytrauma and 3 normal healthy volunteers. The use of human blood samples was approved by the Institutional Review Board of Duke University Medical Center.