Chitinase assays

Culture supernatants were harvested and subjected to chitinase assays according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). β-1,3-glucanase assays were performed in 0.05 M sodium citrate buffer (pH 4.5) with β-1,3-glucan for 2 h. The reaction was stopped by heating at 100 °C for 5 min, and the amount of reducing sugar liberated was measured using neocuproine.

One hundred microliters of mouse mammary gland homogenate were mixed with 300 µl lysis buffer supplemented with protease inhibitors (200 mM NaCl, 10 mM Tris–HCl pH 7.4, 5 mM EDTA, 1% Nonidet P-40, 10% glycerol, 1 mM oxidized l-glutathion, 100 µM PMSF, 2.1 µM leupeptin, and 0.15 µM aprotinin) to extract cellular proteins. The suspensions rested overnight at −20°C, were centrifuged at 12,250 g for 1 h and finally the supernatant was centrifuged for another 30 min to precipitate the pellet. Protein concentration of the lysate samples was determined as previously described (36 (link)). Cytokine quantification (ProcartaPlex from eBioscience), chitinase assays (Sigma), and CHI3L1 ELISA (Biotechne) on the mammary gland lysates were all performed according to the manufacturer’s instructions. Briefly, chitinase activity assay determines exo- and endochitinase activity through the production of fluorogenic products. The exochitinase activity releases one N-acetyl-d-glucosamine residue from the (non)reducing end, and can be measured with an artificial substrate such as 4-methylumbelliferyl (MU) N,N′-diacetyl-β-d-chitobiose, while the endochitinase activity prefers longer N-acetyl-d-glucosamine chains and can, therefore, be measured with substrates such as 4-MU N,N′,N′′-triacetyl-β-d-chitotriose (11 (link)).