MSU crystals were prepared as described previously (2 (link)). 1 g of uric acid (Sigma) in 180 ml 0.01 M NaOH was heated to 70° C. NaOH was added as required to maintain pH between 7.1 and 7.2 and the solution was filtered and incubated at RT with slow and continuous stirring for 24 h. MSU crystals were kept sterile, washed with ethanol, dried, autoclaved and re-suspended in PBS by sonication. MSU crystals contained < 0.005 EU/mL endotoxin (LAL endotoxin assay, GenScript).
In most experiments (and unless stated otherwise), we injected 0.5 mg MSU i.a. in 10 μl PBS in one ankle and PBS only in the contra-lateral ankle. We used Microliter Syringes #705 (Hamilton) with 27G needles for all i.a. injections. Injections were performed under isoflurane anesthesia, and the quality of i.a. injection was controlled by assessing location of MSU crystals deposition by histology on ankle tissue collected 24 h after the injection. In some experiments, we used MC-deficient mice engrafted with WT BMCMCs in one ankle and IL-1β−/− BMCMCs in the contra-lateral ankle and injected these mice with MSU crystals in both ankles (Figure 3, A and B ). We also injected DT-treated Cpa3-Cre; iDTR mice with MSU crystals in both ankles (Figure 4, F and G ). Ankle swelling was measured at different time points using a precision caliper (Fisherbrand Traceable Digital Calipers; Fischer Scientific).
In most experiments (and unless stated otherwise), we injected 0.5 mg MSU i.a. in 10 μl PBS in one ankle and PBS only in the contra-lateral ankle. We used Microliter Syringes #705 (Hamilton) with 27G needles for all i.a. injections. Injections were performed under isoflurane anesthesia, and the quality of i.a. injection was controlled by assessing location of MSU crystals deposition by histology on ankle tissue collected 24 h after the injection. In some experiments, we used MC-deficient mice engrafted with WT BMCMCs in one ankle and IL-1β−/− BMCMCs in the contra-lateral ankle and injected these mice with MSU crystals in both ankles (