Human mpo

Neutrophil infiltration in pancreas and lung tissues was assessed by measuring myeloperoxidase (MPO) activity as previously described (Lugea, et al., 2017b (link)). Tissue samples were homogenized in 20 mmol/L potassium phosphate buffer (pH 7.4) containing 1 mmol/L EDTA, and thereafter resuspended in 50 mmol/L potassium phosphate buffer (pH 6) containing 0.5 % hexadecyltrimethyl ammonium bromide. The samples were then processed by three freeze-thaw cycles, sonicated at 60°C for 2 h, and centrifuged for 20 min at 14,000 g. MPO activity was measured in aliquots of the supernatants mixed with 3,3,5,5-tetramethylbenzidine as substrate for MPO (final concentration 1.6 mmol/L) diluted in 80 mmol/L potassium phosphate buffer (pH 5.4) containing freshly added H₂O₂ (final concentration 3 mmol/L). Absorbance at 620 nm was measured for 3 min at 30 sec intervals. MPO activity was estimated by comparison of values to those obtained with known concentrations of human MPO (Sigma Chemical, St. Louis, MO) and expressed as activity units per tissue weight.

Human MPO, iron-free Ent, ferric chloride, hydrogen peroxide, O-dianisidine hydrochloride, DHBA, ABAH, dimethyl sulfoxide, ferrichrome and 2,2^’ dipyridyl were procured from Sigma (St Louis, MO). Yersiniabactin (ybt) and salmochelin S4 were obtained from EMC microcollections (GmbH, Germany). Bovine milk LPO was purchased from Worthington Biochemical Corp., (Lakewood, NJ). Guaiacol (2-methoxyphenol) was obtained from Alfa Aesar (MA, USA). Human Lcn2 (also known as neutrophil gelatinase-associated lipocalin), murine Lcn2 (also known as 24p3) and Human MPO were purchased from R&D Systems (Minneapolis, MN). The commercially available recombinant Lcn2 proteins were free of endotoxin, siderophore and iron.