Arginase Activity Determination Protocol

J774 cells (1x10⁶/ml) or peritoneal cells (1x10⁶/ml) were treated as previously described above. Arginase activity was measured in cell lysates as described previously 16 (link), 39 (link). Briefly, cells were lysed with 50 µl of 0.1% Triton X-100 containing protease inhibitors. This mixture was stirred for 30 min and then 50 μl of 10 mM MnCl2 with 50 mM Tris-HCl we added to activate the enzyme for 10 min at 56 ◦C. Arginine hydrolysis was initiated by the addition of 25 µl of 0.5 M L-arginine, pH 9.7, at 37 ◦C for 45 min. The reaction was stopped with a mixture of acids, and the urea concentration was measured at 540 nm after the addition of 25 μl of α-isonitrosopropiophenone (dissolved in 100% ethanol) followed by heating at 95 ◦C for 45 min. The results are expressed as Arginase Index (fold increase of arginase activity in samples above basal).