Female Balb c mice 9 to 12 weeks of age were obtained from the Jackson Laboratory. All mice used in this study were main tained under standard conditions at the Stanford Uni versity Research Animal Facility. NETs from product info the EPRO cell line were prepared as described below. NETs were prepared and stored at a concentration of 10 ug ml of which 200 ul was subcutaneously injected at each immunization. Each treatment group included 3 mice, immunized weekly over 4 weeks with NETs alone, or NETs com bined with murine cathelicidin related antimicrobial peptide at a 5,1 w w ratio to NETs. Pro teinuria was assessed by dipstick analysis using Albustix. Inhibitors,Modulators,Libraries Mouse serum samples Inhibitors,Modulators,Libraries were obtained by saphenous vein bleeding immediately before the first injection and once every 4 weeks after immuni zation for up to 12 weeks.
Cell culture and differentiation Inhibitors,Modulators,Libraries of neutrophils Maintenance and differentiation of myeloid cell lines into neutrophils were performed at 37 C and 5% carbon dioxide as previously described. 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 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 Inhibitors,Modulators,Libraries media supplemented with 20% horse serum, 2 mM L glutamine, 1X P S, and 15% stem cell factor Inhibitors,Modulators,Libraries 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 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 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 selleckchem 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. Tsai and main tained at 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, and differentiated for 3 days into neutrophils from a starting density of 3 105 cells ml with a final concentration of 10 uM ATRA.