Arginase assay

BMDMs were washed twice and lysed in 10 mM Tris–HCl pH 7.4 with protease inhibitors and 1% Triton X-100 for 30 min on ice with vigorous vortexing every 10 min. The samples were centrifuged at 13,000g for 20 min to remove debris. Protein content was normalized. Arginase activity was assayed using an Arginase Activity Assay Kit (Sigma). Briefly, 40 μL of cell lysate was added to each well in quadruplicate. 1 mM urea and ddH₂O were used as a standard and standard blank. 10 μl substrate buffer was added to each sample well and each sample blank well received no addition. Plates were sealed and incubated at 37 °C for 2 h. Urea reagent (200 μl) was added to all wells to stop the reaction and substrate buffer (10 μl) was added to the sample blank wells. Plates were incubated at RT for 60 min. If turbidity appeared in the wells, the plate was centrifuged and the supernatants were transferred to a new plate without agitation of the precipitate. The absorbance was read at 430 nm using a SpectraMax M2 (Molecular Devices).