Laboratory Animals published by the US National Institutes of Health. Anesthetic methods to minimize suffering as much as possible are detailed in the Methods. isolated as described above. Of note, all tubes were treated with Sigmacote to prevent adherence of fibroblasts to the plastic tubes during isolation. After isolation, cells were plated in fibroblast growth medium. Cells were washed and medium replaced on Day 1. On day 4, cells were about 80% confluent and medium exchanged for macrophage conditioned media as described below. Luminex Assay Conditioned media was collected from bone marrow derived macrophages or cardiac explant cultures as previously described. Media was incubated with beads from a mouse 20-plex cytokine detection kit in a 96 well plate. After addition of secondary antibodies, bound cytokine concentrations were measured using a Luminex plate reader. Standard curves were run for all cytokines and in the case of explant tissue media, values normalized to tissue weight. Experimental Animals Generation of SR-uPA+/0 mice has been previously described. In this paper all mice described are of the high expressing M87 line. The SR-uPA+/o mice were backcrossed into the C57BL/6 background for at least 8 generations then bred with nontransgenic C57BL/6 littermates to obtain experimental and control mice. Some mice were bred with mice deficient 9726632 in IL-6 to generate IL-6+/2 mice with and without the SR-uPA transgene. Littermates were bred to generate experimental mice. Taqman Quantitative PCR RNA was isolated using RNAeasy. RNA was processed using the Thermoscientific Verso 1 stem RT-qPCR kit according to the manufacturer’s instructions. Pre-validated primer/probe sets were obtained from Applied Biosystems. All PCR reactions were normalized internally using either an eukaryotic 18 S RNA primer set or GAPDH RNA primer set as an endogenous control and run in triplicate. Quantitative Taqman PCR was performed using the Applied Biosystem 7900HT Real Time PCR System and analyzed using the Applied Biosystem 7900HT System sequence detection software, version 2.3. Histologic Analyses At appropriate time-points, mice were deeply anesthetized with intra-peritoneal ketamine and xylazine. Anesthesia was deemed adequate when mice no longer responded to foot pinch. Hearts were excised, placed in PBS, transferred to PBS with 5% dextrose and 25 mmol/L KCl to produce cardiac arrest, weighed and then placed in sucrose formalin fixative. Hearts were sectioned into three pieces and processed into a single paraffin block. Macrophages were detected with a rat monoclonal antibody and quantified as previously described. Collagen accumulation was quantified by picrosirius red staining of a single section from the mid-ventricle of each heart as previously described. Quantification of cardiac macrophages and collagen was done by observers AZD-0530 site blinded to genotype and treatment. Statistical Analysis 1700309 Data that are normally distributed are presented as bar graphs with mean 6 S.D. and are compared with Student’s t-test. Data that were not normally distributed are presented as dot plots with median. Group medians are compared with the Macrophage Phenotype in Cardiac Fibrosis Mann-Whitney rank-sum test. Some data not normally distributed is presented in figures with mean 6 S.E.M. for clarity. with our previous data in wild-type mice, NTG littermates had only rare Mac-3 positive cells in both the Il6+/2 and Il62/2 backgrounds. Results The pro-inflammatory Cytokine IL-6
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