Types. Our results also indicate that EPICs display a characteristic mobilization and proteolytic program, a finding that is relevant to our knowledge of the structure of adult tert-Butylhydroquinone cardiac interstitium, the definition of a cardiac stem cell niche and the interstitial response to stress or damage. This work opens new avenues for the study of cardiac fibroblast/myofibroblast biology and the analysis of mechanisms leading to cardiac remodeling of the GHRH (1-29) supplier diseased heart.epicardial cells attached to the substrate. These epicardial cells were cultured for an extra period of 48 hours.Generation of the EPIC cell lineThe EPIC is a continuous cell line derived from E11.5 mouse embryonic epicardium generated at the University of Malaga ?(Spain). These cells were primarily extracted from whole E11.5 embryonic hearts as described above. Epicardial cell monolayers were cultured for 24 extra hours in DMEM, Penicillin/Streptomycin (GIBCO) and 1 mg/ml 20-methylcholanthrene (MCA, SIGMA). Purity of primary cultures was assessed by cytokeratin immunostaining (see below). After extensive washing with DMEM, cells were let to grow for 4 weeks in their wells, with new DMEM, 1 fetal bovine serum (FBS, PAA) and Penicillin/ Streptomycin added every two days. After confluence, cells were trypsinized, replated and cultured at high concentration in DMEM, 10 FBS and Penicillin/Streptomycin. The EPIC line has been growing in culture for more than 3 years.EPIC culture dynamicsFor regular culture, EPIC were maintained in high glucose DMEM supplemented with 10 FBS, 100 U/mL of penicillin, 100 mg/mL streptomycin and 25 mg/mL of plasmocin (INVIVOGEN), and routinely passaged at confluence. To plot the growth curve, 104 cells were plated in 100 mm diameter Petri dishes for 10 days; each day 3 dishes were trypsinized and the number of cells was estimated from the suspension in a Neubauer chamber.Differentiation assaysTo promote the differentiation of embryonic epicardial progenitors (E9.5 proepicardial cells), E11.5 epicardial cells and EPIC (1.66104 cells/well), samples were cultured in high glucose DMEM supplemented with 1 FBS, 100 U/mL of penicillin and 100 mg/mL streptomycin for 24 hours. All samples were cultured for 24 extra hours in two different media conditioned to promote cell type-specific differentiation: 5 FBS for smooth and cardiac striated muscle; 50 ng/mL bFGF (R D)+100 ng/mL VEGF-A (R D) for vascular endothelium.Immunohistochemical characterization Materials and Methods Ethics statementThe research on mouse embryonic tissue carried out in this study has been approved by the Ethics Committee of the University of Malaga (Spain) under a specific procedure for the ?controlled breeding of mice and embryo collection. All the work performed in this study was developed in compliance with the Spanish (LAW 32/2007; RD142/2002; RD1201/2005) and European regulations (Directive 86/609/EEC; Directive 2010/ 63/EU; Commission Recommendation 2007/526/EC) on the use of animals for scientific research. Cells were fixed in 70 methanol, Dent’s fixative (methanol:DMSO, 4:1) or 4 paraformaldehyde, hydrated through a 70 , 50 , 30 ethanol series, extensively washed in PBS, permeabilized and blocked in 5 normal goat serum, 1 bovine serum albumin (BSA) and 0.5 Triton X-100 in Tris-PBS (SBT). Then, cells were incubated overnight in the primary antibody diluted in SBT [1:100 a-SMA (SIGMA); 1:20 MF20 (DSHB); 1:50 SM22 (SIGMA); 1:100 smooth muscle myosin (Biomedical Technologies); 1:100.Types. Our results also indicate that EPICs display a characteristic mobilization and proteolytic program, a finding that is relevant to our knowledge of the structure of adult cardiac interstitium, the definition of a cardiac stem cell niche and the interstitial response to stress or damage. This work opens new avenues for the study of cardiac fibroblast/myofibroblast biology and the analysis of mechanisms leading to cardiac remodeling of the diseased heart.epicardial cells attached to the substrate. These epicardial cells were cultured for an extra period of 48 hours.Generation of the EPIC cell lineThe EPIC is a continuous cell line derived from E11.5 mouse embryonic epicardium generated at the University of Malaga ?(Spain). These cells were primarily extracted from whole E11.5 embryonic hearts as described above. Epicardial cell monolayers were cultured for 24 extra hours in DMEM, Penicillin/Streptomycin (GIBCO) and 1 mg/ml 20-methylcholanthrene (MCA, SIGMA). Purity of primary cultures was assessed by cytokeratin immunostaining (see below). After extensive washing with DMEM, cells were let to grow for 4 weeks in their wells, with new DMEM, 1 fetal bovine serum (FBS, PAA) and Penicillin/ Streptomycin added every two days. After confluence, cells were trypsinized, replated and cultured at high concentration in DMEM, 10 FBS and Penicillin/Streptomycin. The EPIC line has been growing in culture for more than 3 years.EPIC culture dynamicsFor regular culture, EPIC were maintained in high glucose DMEM supplemented with 10 FBS, 100 U/mL of penicillin, 100 mg/mL streptomycin and 25 mg/mL of plasmocin (INVIVOGEN), and routinely passaged at confluence. To plot the growth curve, 104 cells were plated in 100 mm diameter Petri dishes for 10 days; each day 3 dishes were trypsinized and the number of cells was estimated from the suspension in a Neubauer chamber.Differentiation assaysTo promote the differentiation of embryonic epicardial progenitors (E9.5 proepicardial cells), E11.5 epicardial cells and EPIC (1.66104 cells/well), samples were cultured in high glucose DMEM supplemented with 1 FBS, 100 U/mL of penicillin and 100 mg/mL streptomycin for 24 hours. All samples were cultured for 24 extra hours in two different media conditioned to promote cell type-specific differentiation: 5 FBS for smooth and cardiac striated muscle; 50 ng/mL bFGF (R D)+100 ng/mL VEGF-A (R D) for vascular endothelium.Immunohistochemical characterization Materials and Methods Ethics statementThe research on mouse embryonic tissue carried out in this study has been approved by the Ethics Committee of the University of Malaga (Spain) under a specific procedure for the ?controlled breeding of mice and embryo collection. All the work performed in this study was developed in compliance with the Spanish (LAW 32/2007; RD142/2002; RD1201/2005) and European regulations (Directive 86/609/EEC; Directive 2010/ 63/EU; Commission Recommendation 2007/526/EC) on the use of animals for scientific research. Cells were fixed in 70 methanol, Dent’s fixative (methanol:DMSO, 4:1) or 4 paraformaldehyde, hydrated through a 70 , 50 , 30 ethanol series, extensively washed in PBS, permeabilized and blocked in 5 normal goat serum, 1 bovine serum albumin (BSA) and 0.5 Triton X-100 in Tris-PBS (SBT). Then, cells were incubated overnight in the primary antibody diluted in SBT [1:100 a-SMA (SIGMA); 1:20 MF20 (DSHB); 1:50 SM22 (SIGMA); 1:100 smooth muscle myosin (Biomedical Technologies); 1:100.