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An informed FARP1 consent was obtained from all patients or their relatives. In stroke patients, blood samples were obtained on day 1, 3 and 12 of hospital stay. Lumbar puncture was performed twice, i.e. on day 3 and 12 of disease onset. Control group patients underwent single CSF and blood sampling for biochemistry parameters of apoptosis. Blood samples were allowed to clot at room temperature for 30 min and centrifuged at 3000 g for 10 min. Serum and CSF samples were aliquoted and stored at ?20��C until analysis. sFas/APO 1 levels in serum and CSF samples were determined by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Quantikine Human sFas Immunoassay, R&D Selleck Obeticholic Acid Systems, Minneapolis, USA) according to the manufacturer's instructions. Briefly, sFas/APO 1 standards, 10-fold diluted serum and undiluted CSF samples were pipetted into the wells coated with murine monoclonal antibody to human sFas/APO 1. After two hours of incubation, unbound substances were washed away and horseradish peroxidase conjugated polyclonal antibody against Fas/APO 1 was added to the wells. Following two hours of incubation and a wash, tetramethylbenzidine substrate solution was added to the wells. Color developed in proportion to the amount of sFas/APO 1 bound in the initial step. The color development was stopped after 30 minutes with sulfuric acid and the intensity of color was measured using a microplate reader set to 450 nm with wavelength correction at 540 nm. Serum and CSF sFas/APO 1 levels were calculated from a standard curve generated by plotting the mean optical density for each standard concentration on the y-axis against the sFas/APO 1 concentration on the x-axis. Statistical analyses were performed using the commercial system SAS System for Windows, Version 9.3. Normality of distribution of the variables was tested with the Kolmogorov-Smirnov test. The results are expressed by mean value and standard deviation while non-normally distributed variables are expressed by median and interquartile range (IQR). Differences in serum and CSF parameters between stroke patients and control group were assessed by Kruskal-Wallis test, followed by Mann-Whitney U-test with P value adjustment, Doxorubicin in vitro i.e. reducing the level of significance depending on the number of comparisons (P 0.05/2=0.025 and P 0.05/3=0.017). Within-group differences were assessed by Wilcoxon rank-sum test on comparison of two dependent samples (analysis of difference in CSF parameters), whereas Friedman two-way analysis of variance by ranks was used on comparison of multiple dependent samples (analysis of difference in serum parameters). Correlations between particular variables were tested by Spearman rank correlation. Statistical significance was set at P
