The amplification was performed in CFX96 Real-time thermocycler (Biorad Laboratories, Hercules, CA, USA) as previously described [17]. Viability of A498 cells stimulated with E. coli The A498 cell line was stimulated with the different bacterial isolates and the viability of the cells was assessed after 6 h. Multiplicity of infection (MOI) of 10 was used (5 · 105 cells were stimulated with 5 · 106 CFU of bacteria).

The viability of the cells was assessed by the trypan blue (0.4%) exclusion test in a cell counter (TC10™ automated cell counter, Bio-Rad) and by the cytotoxicity detection kit plus-LDH (Roche Diagnostics, Indianapolis, IN, USA) according to manufacturer’s protocol. Isolation of polymorphonucleated leukocytes Human polymorphonucleated leukocytes (PMN) were isolated from whole blood SBI-0206965 molecular weight using polymorphprep (Axis-Shield PoC AS, Oslo, Norway). Blood was collected according to the swedish national board of health and https://www.selleckchem.com/products/VX-765.html welfares guidelines and the ethical guidelines of the declaration of Helsinki. The healthy volunteers gave a written Luminespib concentration informed consent for research use and the samples were anonymized immediately after collection. The donors were not subjected to extra harm or risk as the blood was collected at the same occasion as a blood donation. According to paragraph 4 of the swedish law (2003:460) on ethical conduct in human research, this study did not require

ethical approval. Briefly, polymorphprep was layered with an equal volume of heparinized blood and centrifuged at 1350 rpm for 40 min at room temperature. The PMN fraction was collected and an equal volume of 0.45% NaCl and 20 ml PBS was added. Any remaining erythrocytes were removed by hypotonic lysis with sterile milliQ water. Cold PBS containing 3.4% NaCl and Krebs-Ringer glucose Carteolol HCl (KRG) were added to restore osmotic pressure. The PMN were centrifuged, the supernatant discarded and the pellet resuspended in 1 ml PBS, KRG + Ca2+ or DMEM + 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The viability of the PMN was > 90% as determined by the trypan

blue exclusion test. Measurement of total ROS-production Total reactive oxygen species (ROS)-production of PMN was measured with a luminol-horseradish peroxidase (HRP) assay. Luminol is activated by H2O2 and the evoked luminescence is proportional to ROS-production. PMN in KRG + Ca2+ were incubated with luminol (0.1 mg/ml, Sigma) and HRP (4 U/ml, Roche) for 15 min at 5% CO2 and 37°C. PMN and bacteria (MOI 10) were combined in a 96-well plate. Phorbol-12-myristat-13-acetat (PMA) was used as positive control and KRG + Ca2+ as negative control. The plate was centrifuged at 400 × g at 4°C for 3 min and the luminescence was measured in a microplate reader (Fluostar Optima, BMG Labtech, Aylesbury, UK) every third min for 4 h. All samples were run in duplicate.