Rhodamine B isothiocyanate was covalently conju gated with each chitosan to generate either RITC 80 M or RITC 95 M. The DDA of RITC chitosan derivatives is reported as unchanged, given that the derivatization level was determined selleckchem as only 0. 5% mol RITC mol chitosan. Ficoll Paque and dextran used for the isolation Inhibitors,Modulators,Libraries of PMN were obtained from Pharmacia and fetal bovine serum as well as RPMI 1640 were purchased from Wisent. Calcein AM was obtained from Invitrogen. Migration was assessed using the ChemoTx system from Neuroprobe and the purified myeloperoxidase, O dianisidine dihy drochloride, hydrogen peroxide, and cytochalasin B used in the MPO assay were obtained from Sigma Aldrich. Hexadecyltrimethylammonium bromide was purchased from Fluka Chemie GmbH. Cytochrome c equine heart and pyrrolidine 1 were purchased from Calbiochem.

Isolation Inhibitors,Modulators,Libraries of polymophonuclear neutrophils The Institutional Review Board of the Universit�� Laval approved the study, and volunteers signed a consent form. PMNs were isolated as previously described. Briefly, venous blood was obtained from healthy adult volunteers, in accordance with institute approved protocols, in tubes containing heparin or isocitrate. No Inhibitors,Modulators,Libraries difference was observed between the results obtained with these two anticoagulants. After sedimentation of red blood cells in 2% dextran, PMNs were aseptically purified by centrifugation on Ficoll Paque cushions. Contaminating eryth rocytes were removed by hypotonic lysis and PMNs were resuspended in RPMI 1640 supplemented with 0. 1% FBS previously decomplemented at 56 C for 30 minutes, except for the chemotaxis experiment.

Chemotaxis Chemotaxis was measured as described previously. Briefly, PMNs were resuspended in RPMI 1640 and 10% FBS at a concentration of 107 cells ml and were pre incubated with 1 g ml calcein AM at 37 C for 30 minutes in the dark with constant agitation. Cells were washed twice and resuspended in RPMI 10% FBS at 5 107 cells ml at 37 C. PMN migration was Inhibitors,Modulators,Libraries monitored using a 96 well ChemoTx disposable chemo taxis system. The fluorescence of cells in the filters was meas ured using a microplate fluorescence reader. Fluorescence was con verted to numbers of PMNs based on a standard curve gener ated by seeding known numbers of PMNs in the bottom of the chamber. The results are expressed as percentage of migrated cells, calculated as the fluorescence of migrated PMNs fluorescence of 20,000 PMNs ml 100, obtained from the standard curve.

In some experiments, PMNs were incu bated with a final concentration of 0. 5 g ml pertussis toxin for 90 minutes or with 10 7 mol l pyrrolidine 1 for 10 minutes Inhibitors,Modulators,Libraries at 37 C before the chemotaxis selleckchem Imatinib Mesylate assay. Production of superoxide anions by polymorphonuclear neutrophils Superoxide anion production in response to 80 M and 95 M chitosan was determined using the cytochrome c reduction assay, as previously described.