E: Representative Sytox green extracellular DNA fluorescence and phase-contrast images of neutrophils used for NET quantification. IRI. Moreover, the rAAV8-TIMP-1 vector therapy enhanced significantly the 7-day survival rate of without significant toxicity or immune response.21, 22 Materials and Methods Mice and Model of Hepatic IRI C57BL/6 mice and C57BL/6 mice carrying the null allele (B6.129S4-biodistribution analysis and 1 week before surgery for hepatic IRI studies, including the 7-day mouse survival study. AAV8 Antibody Detection in Mouse Serum Previous exposure to AAV8 viral capsid was assessed in mouse serum samples by enzyme-linked immunosorbent assay.25 Enzyme-linked immunosorbent assay plates were coated with 1??109 genome copies AAV8 per well. Positive control serum samples were collected from AAV8 injected mice with established anti-AAV8 antibody titers. Assessment of Liver Damage Liver specimens were fixed in 10% buffered formalin, embedded in paraffin, and processed for hematoxylin and eosin and Sirius Penicillin V potassium salt Red staining, as previously described.26 Serum alanine Penicillin V potassium salt transaminase and aspartate transaminase levels were evaluated using a commercial kit (Teco Diagnostics, Anaheim, CA). Isolation and Culture of Mouse Cells Isolation of neutrophils and hepatocytes was performed according to previously published methods.3, 27 Briefly, to isolate neutrophils, bone marrow was flushed from femurs and tibias (stripped of all muscle and sinew) with 2.5 mL of RPMI-1640 medium containing 5% fetal calf serum on ice. Hypotonic lysis was used to remove erythrocytes. The bone marrow flush was resuspended in Hanks’ balanced saline solution, layered on a Percoll (Sigma-Aldrich, St. Louis, MO) gradient (3 mL of 55%, top; 3 mL of 65%, middle; 4 mL of 80% Percoll), and centrifuged at 872 for 30 minutes at 10C. Mature neutrophils were collected from the interface of the 65% and 80% fractions. The neutrophil-rich fractions were 90% pure (Ly-6G immunostaining/morphology) and 95% Penicillin V potassium salt viable (trypan blue exclusion). To isolate hepatocytes, anesthetized Penicillin V potassium salt mice were subject to a midline laparotomy and cannulation of the inferior vena cava for liver perfusion with EDTA chelating and collagenase perfusion buffers. Hepatocytes were separated from nonparenchymal cells by successive low-speed centrifugation steps and resuspended in Williams E Medium with primary hepatocyte IL9 antibody maintenance supplements (Life Technologies, Carlsbad, CA). Immunoperoxidase and Immunofluorescence Assays Acetone-fixed cryostat liver sections or 4% paraformaldehyde fixed neutrophils were incubated with antiCLy-6G (1A8; BD Biosciences, San Jose, CA), antimacrophage-1 antigen (Mac-1 M1/70; BD Biosciences), antiCMMP-9 (AF909; R&D Systems, Minneapolis, MN), antiCTIMP-1 (AF980; R&D Systems), and antiCcitrullinated histone H3 (Cit-H3; Abcam, Cambridge, MA) antibodies, as described.13 The sections were evaluated blindly (S.D.) by counting 10 high-power fields/section in triplicate. Dual/triple staining was detected by immunofluorescence with Alexa Fluor 594-red anti-rabbit IgG and anti-goat IgG (H+L), Alexa Fluor 488-green anti-rat IgG (H+L), Alexa Fluor 350-red anti-rabbit IgG (H+L), and Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific, Waltham, MA). Slides were analyzed under a Penicillin V potassium salt Nikon Eclipse 90i Fluorescence Microscope (Nikon, Melville, NY). Gelatin Zymography Analysis Liver protein extraction and zymography analyses were performed as previously described.7 Gelatinolytic activity was detected in liver extracts (100 g final protein content) using 10% SDS-PAGE gels containing 1 mg/mL of gelatin (Thermo Fisher Scientific) under nonreducing conditions. Recombinant MMPs (BIOMOL International, Plymouth, PA) and prestained molecular weight markers (Bio-Rad Laboratories, Hercules, CA) served as standards. Zymography Cellular localization of gelatinolytic activity was examined on frozen and unfixed liver cryosections by zymography, as previously described.28 First, MMP-9 protein was immunofluorescently stained with goat antiCMMP-9 (AF909; R&D Systems) primary antibody and an Alexa Fluor 594 anti-goat IgG (H+L) (Thermo Fisher Scientific). Subsequently, quenched fluorogenic DQ-gelatin substrate (Thermo Fisher Scientific) was dissolved at 1 mg/mL in zymogram developing buffer (diluted, 1:10; Thermo Fisher Scientific) and then diluted 1:10 in Agarose type VII-A, low gelling temperature (1% w/v; Sigma-Aldrich). Fifty microliters of DQ-gelatin solution in agarose was.