Glycosaminoglycans (GAGs) are linear negatively charged polysaccharides and important components of extracellular matrices and cell surface area glycan layers like the endothelial glycocalyx. 1,9-dimethylmethylene blue (DMMB) assays, leading to an overestimation of GAG produces. We hypothesized that RNA may be contaminating GAG ingredients from various other cell civilizations and perhaps tissues, and therefore looked into potential RNA contaminations in GAG ingredients from two extra cell lines, individual umbilical vein endothelial cells and retinal pigmental epithelial cells, and mouse kidney, liver organ, heart and spleen tissue. GAG ingredients from all analyzed cell lines and tissue contained varying levels of contaminating RNA, which interfered with GAG quantification using DMMB assays and characterization of GAGs by barium acetate gel electrophoresis. We as a result recommend routinely analyzing the RNA articles of GAG ingredients and propose a sturdy process for GAG isolation which includes an RNA digestive function step. Launch Glycosaminoglycans (GAGs) are linear, adversely charged polysaccharides and prominent the different parts of extracellular cell and matrices surface glycan layers. GAGs are synthesized from duplicating disaccharide blocks 571203-78-6 & most Rabbit Polyclonal to NDUFA9 GAGs, including heparan sulfate (HS), heparin, keratan sulfate, chondroitin sulfate (CS) and dermatan sulfate (DS), could be improved by sulfation, which makes them strongly billed negatively. For instance, HS includes uronic and N-acetylglucosamine acidity disaccharide blocks and may become sulfated in the N-, 2O-, 3O- and 6O-positions from the carbohydrate band structures. The series of adjustments along the carbohydrate backbone enables sulfated GAGs, hS particularly, to 571203-78-6 bind development elements, chemokines and mobile adhesion molecules, such as for example fibroblast growth elements, interleukin-8, selectins as well as the macrophage-1 antigen (Mac pc-1), regulating different physiological procedures including cell development therefore, morphogenesis, inflammation and coagulation [1C9]. GAG manifestation and modifications tend to be cells- and cell type-specific [10, 11]. Consequently, isolation and characterization of GAGs from different cells or cell ethnicities is vital that you unravel cells- and cell type-specific GAG framework and function [12]. Previously, we’ve isolated and characterized a distinctive mouse glomerular endothelial cell range (mGEnC-1) [13] and determined particularly sulfated HS domains in the glomerular endothelial glycocalyx that mediate chemokine binding and leukocyte trafficking during swelling and [13C18]. Because the presence of several additional practical GAG domains in the glomerular endothelial glycocalyx can be presumed, isolation and evaluation of undamaged GAGs, e.g. using mass spectrometry, may yield novel structural information about functional GAG domains. Described GAG extraction protocols usually involve release of GAGs using chaotropic buffers, non-ionic detergents, protease treatment or alkaline -elimination, 571203-78-6 followed by removal of contaminants by enzymatic or chemical digestion, selective precipitation or chromatography [12, 19C21]. GAG quantification in mGEnC-1 GAG extracts initially suggested high yields, with HS as the major component of the mGEnC-1 glycocalyx, as was previously described [22]. However, the obtained HS fraction appeared largely resistant to digestion with bacterial heparinases I, II and III, suggesting that the sugars which co-migrated with HS standards during barium acetate agarose gel electrophoresis contained non-HS compounds. Subsequently we identified RNA as a major contaminant. Here, we describe a GAG isolation protocol including an RNAse treatment that yields GAG extracts that can be reliably visualized by agarose gel electrophoresis and quantified by the DMMB method. Materials and methods Cell culture and animal tissue Conditionally immortalized mouse glomerular endothelial cells (mGEnC-1) were cultured as previously described [13]. Briefly, mGEnC-1 were grown at the proliferative temperature of 33C in 1% gelatin (Sigma-Aldrich)-coated culture flasks (Corning Life Sciences) with DMEM/Hams F12 medium (3:1; Life Technologies) supplemented with 5% fetal bovine serum (FBS; Bodinco), 1% penicillin/streptomycin (PS; Life Technologies) and 20 units (U)/ml recombinant mouse interferon- (IFN-; PeproTech). For differentiation, mGEnC-1 were seeded at 25% density in uncoated culture flasks and cultured for seven days in DMEM/Hams F12 without IFN- in the nonpermissive temp of 37C. Major human being umbilical vein endothelial cells (HUVEC) had been expanded to confluence on 1 g/cm2 bovine fibronectin (Bio-Connect)-covered tradition flasks in endothelial cell development moderate (EGM)-2 (Lonza). Immortalized retinal pigmental epithelial cells.