Polymorphonuclear Apoptosis Compound Library order granulocytes (PMN) were isolated using a

blood lysis method. One hundred microliter aliquots of whole blood were placed in flow cytometry tubes and 1 mL of lysis solution (containing <15 mL formaldehyde and <50 mL diethylene glycol; Becton Dickinson, UK) was added to each tube at room temperature for 15 minutes. The solution was centrifuged at 1,600 rpm for 5 minutes at 18°C and the supernatant discarded leaving the PMN pellet at the bottom of the tube. Twenty microliters of each of two antibody stains (antihuman CD16-phycoerythrin(PE)IgG1κ and anti-human CD11b-APCCy7IgG1κ; Becton Dickinson, UK) was then added and incubated at room temperature in darkness for 25 minutes. The cells were then washed twice with sterile phosphate-buffered saline (PBS) prior to analysis by fluorescence activated cell sorting (FACS)

using a FACS Canto II analyzer and FACS Diva 6.0 software (Becton Dickinson, San Jose, CA). Neutrophils were gated from the PMNs on forward and side-scatter characteristics and the percentage of CD16/CD11b-positive cells were calculated along with the mean fluorescence intensity (MFI). The neutrophil function studies were performed as described.11 All ex vivo studies were performed in pyrogen-free conditions. Neutrophil function was examined in fresh neutrophils isolated from whole blood to more closely resemble physiological conditions and to prevent neutrophil activation during separation, with all samples being performed in triplicate. Phagocytosis was quantified using the Phagotest (Orpegen Pharma), which uses fluorescein isothiocyanate Mitomycin C concentration (FITC)-labeled opsonized E. coli bacteria and analyzed using flow cytometery. selleck products In brief,

100 μL of heparinized whole blood was mixed with 20 μL of FITC-labeled opsonized E. coli (2 × 107) (opsonized with immunoglobulin and complement of pooled sera) and incubated in a water bath at 37°C for 20 minutes. Fluorescence of bacteria at the cell surface was quenched using ice-cold Trypan blue solution. Red blood cells were lysed and PMNs were washed twice with sterile PBS prior to analysis. Neutrophils were gated on forward and side-scatter characteristics and stained with anti-CD16-Phycoerythrin(PE)IgG1 κ and analyzed by FACS. NPA was expressed as the percentage of neutrophils undergoing phagocytosis along with the MFI. The interassay and intraassay coefficient of variance for triplicate samples were 1.6% and 10.1%, respectively. Neutrophil OB was quantified using the Burstest (Orpegen Pharma) which measures the percentage of phagocytic cells that produce ROS. In brief, 100 μL of heparinized whole blood was incubated for 20 minutes with 20 μL of opsonized E. coli (2 × 107), or without stimulus at 37°C. Neutrophil high burst capacity was assessed by adding 5 μL of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to 100 μL of heparinized whole blood.