Consequently, the plates were washed three times with PBS/Tween and incubated with 50 l standard or 4-fold diluted plasma samples in PBS/Tween/FCS for 1 hour on a shaker at room temperature. plasma samples. Third, heparinase was added to plasma samples after in vivo injection of different heparin doses to test its neutralizing effect. Heparinase neutralized up to a 20 U of heparin/mouse and resulted in accurate APTT and element VIII determinations. Summary These procedures Ammonium Glycyrrhizinate (AMGZ) and reagents for plasma preparation and coagulation screening will improve studies on thrombotic disorders in mice. Background Since the recognition of genes encoding mouse clotting proteins and the creation of a growing number of transgenic mice, the use of mouse models for the study of thrombotic disorders offers gained increasing interest [1]. While strategy for determining fibrin deposition in cells [2] and quantification of thrombus formation [2,3] is readily available, ideal utilization of blood samples from mice remains a poorly explored area. Ammonium Glycyrrhizinate (AMGZ) The availability of appropriate laboratory assays is essential in order to study coagulation activation in mice, comparably to humans. There is, however, a stunning lack of analytical methods to test coagulation activation specifically in mice. For human studies, several commercial assays are available, including assays for thrombin-antithrombin (TAT) complexes to test em in vivo /em coagulation activation [4,5]. When these assays, based on antibody detection of human protein epitopes, are applied on mouse plasma, it is likely that problems can arise depending on the degree of interspecies cross-reactivity, level of sensitivity, and specificity. A practical issue of importance is definitely that, traditionally, study on blood coagulation depends on determination of the activity of coagulation factors inside a plasma sample. For this purpose specific anticoagulants, including sodium citrate or cocktails of substances need to be added to blood samples, in order to prevent em ex lover vivo /em clotting. While actually in humans the collection of appropriate plasma samples is not without practical problems, the collection of plasma from mice is definitely a major technical challenge. As far as we know, you will find no published methods for anticoagulation that specifically address plasma sampling in mouse. The use of mouse models enables investigations into the mechanism of coagulation activation in plasma as well as its effects, i.e. fibrin deposition and pathological changes in organs, simultaneously. However, for appropriate cells collection methods such as heparinization are frequently required, which impairs most standard checks of plasmatic coagulation. Human being studies showed that addition of heparinase to plasma samples neutralized up to 2 U/ml heparin resulting in accurate element VIII determinations [6]. In order to minimize interference of heparin we tested the effect of heparinase added to mouse plasma samples following em in vivo /em heparinization. With this study we developed a new mouse specific TAT ELISA and validated this IKBKE antibody method using a mouse model of endotoxemia, characterized by enhanced coagulation activation. Furthermore, we compared this fresh ELISA with two commercially available human being immunoassays for measuring thrombin-antithrombin complexes in mouse plasma samples collected by different blood collection procedures. Methods Animals All studies were performed in male mice of the C57Bl/6 strain (University or college of Maastricht or Academic Medical Center, Amsterdam), which were 8 weeks older and weighing approximately 20 grams at the start of the experiments. The animals were housed Ammonium Glycyrrhizinate (AMGZ) in normal cages in an environment having a 12 hour light-dark cycle, controlled temp (20 2C) and moisture (50 10%). Animals had free access to water and diet programs (Hope Farms, Woerden, The Ammonium Glycyrrhizinate (AMGZ) Netherlands). Endotoxemia studies were performed by i.p. injection of 2 mg lipopolysaccharide (LPS, E. coli, Sigma, St.Louis, MO) per kg. The Experimental Animal Ethics Committee of the University or college Maastricht and of the Academic Medical.