Arginase assay kit

Arginase activity was assessed with an arginase assay kit (Abnova) using an adapted protocol based on the manufacturer’s instructions. Briefly, cell pellets were lysed in 100 μL (10 mM Tris-HCl; pH 7.4) supplemented with protease inhibitor without EDTA (Complete; Roche) and 0.1% Triton X-100. After 30 min of gentle agitation at RT, 5 μL MnCl₂ was added to half of each sample followed by 10 min incubation at 55 °C. MnCl₂ was subsequently added to the control samples. Arginine substrate buffer was added to the MnCl₂-primed samples. After incubation for 2 h at 37 °C, substrate buffer was added to the control samples and urea production was measured by reading the optical density at 430 nm.

Arginase 1 activity in Gr1⁺CD11b⁺cells was determined by measuring urea concentration (a by- product of arginase 1 activity) in the cell lysates using an arginase assay kit (Abnova, Walnut, CA) as described previously [28 ]. One unit of arginase 1 converts 1 μmol of L-arginine to ornithine and urea per minute at pH 9.5 and 37°C.

After the BMDM (5.0 × 10⁶ cells) were stimulated with IL-4 for 48 h, the cells were collected in ice-cold PBS. The cells were then lysed in buffer containing 0.4% Triton-X-100 and protease inhibitors, and the arginase activity were measured using the Arginase Assay Kit (Abnova), according to the manufacturer’s instructions. The lysed cells were centrifuged at 14,000 × g at 4 °C for 10 min, and the supernatants were plated on to a 96-well microtiter plate. L-arginine was converted to urea by a buffer containing a substrate and cofactor, and the absorbance of the samples was measured using a microplate reader at the wavelength of 430 nm.

To detect the arginine uptake in TAM subsets, 5 × 10⁵ TAMs were seeded in a 24-well plate for 24 hours. Then, culture media with or without TAMs were collected to detect the arginine amount with the l-arginine assay kit (BioVision). The levels of arginine uptake were determined by using the amount of arginine in culture medium without TAMs to subtract the amount of arginine in culture medium with TAMs. To detect the arginase activity in TAM subsets, 5 × 10⁵ fresh TAMs were lysed in 10 mM Tris-HCl 7.4 buffer containing 0.4% (w/v) Triton X-100 and protease inhibitors. The arginase activity was measured by the Arginase Assay Kit (Abnova). The lysed cells were centrifuged at 14,000g at 4°C for 10 minutes, and the supernatants were plated onto a 96-well microtiter plate. l-arginine was converted to urea by a buffer containing a substrate and cofactor, and the absorbance of the samples was measured using a microplate reader at the wavelength of 430 nm.

Arginase activity was determined using the arginase assay kit according to the
manufacturer’s instruction (Abnova, Taipei City, Taiwan).