Ated, activated immature, and activated mature platelets, the amount of mice utilised for exactly the same groups have been 16, six, 9 and six, respectively. Platelet-Leukocyte Interactions To investigate platelet-leukocyte interactions in vivo, heparin-anticoagulated blood (50 uL) was incubated with rat anti-mouse CD16/CD32 antibody to block the FcIII/II receptor. A 4-color flow cytometry assay was created to divide the platelet-leukocyte aggregates (PLA, (CD45.2+/CD41+)) into platelet-neutrophil (PNA (CD45.2+/Gr-1+/CD41+)), plateletmonocyte (PMA (CD45.2+/F4/80+/CD41+)), and platelet-lymphocyte (PLyA, estimated as PLA ?(PMA + PNA)) aggregate sub-populations. Rat anti-mouse CD41-APC antibody was employed to label platelets. Saturating concentrations of rat anti-mouse CD45.2-FITC, Gr-1-PE, and F4/80-eFluor450 and isotype controls (eBioscience, San Diego, CA) have been employed for labeling leukocytes. Red blood cells have been lysed with Caltag high-yield lysing remedy (Invitrogen, Camarillo, CA). Neutrophils and monocytes have been distinguished from other cells by their size and granularity. When assessing the proportion of leukocytes involved in PLA formation, CD45.two and CD41 double-positive events have been recorded as a percentage of a total 20,000 gated leukocytes. The percentage of leukocytes forming PLAs was multiplied by the corrected WBC count. For the PLA experiments, the number of mice in every single experimental group had been 12, 6, 12, and 12, respectively. So as to assess the contribution of selectins to colitis-induced PLA formation, some DSS treated mice were treated with the pan-selectin inhibitor fucoidin (30 mg/kg, by way of the femoral vein) at 24 and 12 hrs prior to the flow cytometric measurements.2-Chloropyrimidine-4,5-diamine Order Fucoidan (Sigma-Aldrich, St Louis, MO) was resuspended in endotoxin-free phosphate-buffered saline (PBS).36294-24-3 custom synthesis In this set of experiments, the amount of mice in each group were 12, 12, and 6 for controls, DSS day six untreated, and DSS + Fucoidin.PMID:24856309 PLA formation in these experiments was detected as described above employing 50 uL blood samples derived from the tail vein. Plasma concentrations of thrombopoietin (TPO) and interleukin-6 (IL-6) have been measured in control and DSS treated mice. TPO measurements were obtained working with an ELISA assay, Quantikine mouse Tpo Immunoassay (R D Systems, Minneapolis, MN), whilst a cytokine bead array (BD Biosciences, San Jose, CA) was utilised to determine plasma IL-6 concentration. Data analysis and statisticsNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptThe control values for every single measured variable inside the DSS controls (standard drinking water) didn’t differ substantially over the 6-day period of study. Consequently, the manage values presented in the figures represent the indicates on the 6-day observation period. Information were analyzed applying common statistical analysis. Parametric and non-parametric ANOVA tests had been applied for comparisons of various groups, with all the following post-hoc tests: Dunnett’s (for comparing vs a handle), Newman-Keuls (comparing all groups), and Kruskal-Wallis (non-parametric ANOVA). The Spearman test was utilised for regression analysis. All values are reported as implies ?SE with five mice per group. Statistical significance was set at P 0.05.Inflamm Bowel Dis. Author manuscript; available in PMC 2014 May 01.Yan et al.PageResultsDSS-induced colonic inflammation is linked with thrombocytosis and leukocytosisNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptThere are many published clinical re.