Human myeloperoxidase duoset elisa kit

Complete blood count with differential leukocyte count was assessed using a Sysmex XN-1000™ Hematology Analyzer (Sysmex Group’s, Kobe, Japan). Serum creatinine, BUN, uric acid, total proteins, albumin, bilirubin, C-reactive protein, natrium, potassium, calcium and phosphorus were analyzed using standard laboratory methods (Cobas c501 analyzer, Roche Diagnostic, Basel, Switzerland).
Urinary creatinine was measured using Jaffé reaction as described previously (21 (link)). Briefly, 10 microliters of samples were mixed with 200 microliters of freshly prepared reagent consisted of 0.2 M sodium hydroxide and 25 mM picric acid (5:1 ratio). Absorbance was measured at 492 nm. Absorbance measured after 1st minute was subtracted from the absorbance measured at 6th minute.
Concentration of myeloperoxidase (MPO) in urine was determined using commercially available spectrophotometric assay according to manufactureŕs protocol (Human Myeloperoxidase DuoSet Elisa Kit, R&D Systems, Minneapolis, MN, United States). Absorbance was measured at 450 nm. Limit of detection was 62.5 pg/ml.
Similarly, cathelicidin in urine was measured by commercial Elisa kit as recommended by the manufactureŕs protocol (LL-37 Human Elisa kit, Hycult Biotech, Wayne, PA, United States). Urine samples were 10-times diluted. Absorbance was measure at 450 nm. Limit of detection was 0.14 ng/ml.

Wells of 96-well plates were coated with 30 μg/mL bovine fibronectin (Sigma) overnight at 4°C prior to experiments. Spores were grown in the fibronectin-coated 96-well plates for 8 h at 37°C in GMM until the germling stage (100,000 spores/well in 100 μl). Following germination, GMM was removed and freshly isolated primary human neutrophils were added to wells at a concentration of 200,000 cells/well in 100 μl RPMI + 2% FBS. For every experiment 5 wells were used for each condition. Wells with unstimulated neutrophils were used as a negative control. Supernatants were collected after 3 h of co-incubation and centrifuged for 3 min at 300 x g to pellet any remaining cells. The supernatant was then removed and stored at -80°C. IL-8 was measured using the Human IL-8/CXCL8 DuoSet ELISA kit (R&D Systems) following manufacturer protocol. Supernatants were diluted 1:4 to fit within the standard curve. Myeloperoxidase was measured using the Human Myeloperoxidase DuoSet ELISA kit (R&D Systems) following manufacturer protocol. Supernatants were diluted 1:1000 to fit within the standard curve.