Briefly, the human promyelocyte HL 60 cell line was obtained from ATCC and maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 2 mM L glutamine, 25 mM hydroxyethyl piperazineethanesulfonic acid and 1X penicillin streptomycin. Cells were maintained at a density range of 1 105 to 8 105 cells ml for a maximum of 80 passages, and differ Tipifarnib leukemia entiated for 3 days into neutrophils from a starting den sity of 3 105 cells ml with a final concentration of 1 uM all trans retinoic acid 1. 25% dimethylsulfoxide. The murine multipotent cell line EML was obtained from ATCC and maintained in Iscove��s modified dulbecco��s medium media supplemented with 20% horse serum, 2 mM L glutamine, 1X P S, and 15% stem cell factor containing condi tioned media.

EML cells were maintained in 6 well plates at a density range of 1 105 to 5 105 cells ml and differentiated for two days by adding a final concen tration of 10 uM ATRA and 25 ng Inhibitors,Modulators,Libraries ml recombinant murine IL 3. The cells were further differentiated for one day with fresh media con taining a final concentration of 60 uM ATRA and 150 ng ml recombinant murine IL 3. After washing the cells twice with PBS, they were grown Inhibitors,Modulators,Libraries in IMDM media supplemented with 20% horse serum, 1X P S and 10% BHK HM 5 conditioned medium as a source of GM CSF, for 9 days without splitting cells. At this stage, EML cells had differentiated into EPRO cells, which were maintained in this medium at a density of 0. 5 105 to 8 105 cells ml. EPRO cells were differentiated for 3 days into neutrophils from a starting density of 3 105 cells ml with a final concen tration of 10 uM ATRA.

The murine promyelocyte cell line MPRO was obtained from Dr. Inhibitors,Modulators,Libraries Tsai and main tained at Inhibitors,Modulators,Libraries a density range of 0. 5 105 to 1. 0 106 cells in IMDM media supplemented with 20% horse serum, 1X P S, and 10% BHK HM 5 conditioned medium as a source of GM CSF, Inhibitors,Modulators,Libraries and differentiated for 3 days into neutrophils from a starting density of 3 105 cells ml with a final concentration of 10 uM ATRA. Conditioned media Growth factors required for EPRO and MPRO cultures were obtained using two secreting cell lines and prepared as previously described. Briefly, baby hamster kidney HM 5 cells, which secrete murine GM CSF, or baby hamster kidney MKL cells, which secrete murine SCF, were maintained in DMEM high glucose media supplemen ted with 10% heat inactivated FBS off US origin, 2 mM L glutamine, and 100 U ml penicillin 100 ug ml streptomycin.

These were expanded to T 175 flasks and grown to confluence. Cell culture supernatants SB1518 were harvested when the media turned yel low orange, then sterile filtered and frozen at 20 C until ready for use. Stimulation and isolation of NETs Neutrophils derived from cell lines or isolated from human donors were stimulated to produce NETs as pre viously described. Neutrophils were incubated at 37 C with 5% carbon dioxide, in 100 mm plates or 150 mm plates at 1.