Phosphoprotein phosphatase : An In-depth Analysis On What Works And The things that Doesn't

Each experiment was repeated two times. Percentage of DNA in the tail (% tail DNA) was analyzed. It is positively correlated with the level of DNA breakage or/and alkali labile sites in the cell. The mean value of the % tail DNA in a particular sample was taken as an index of DNA damage in this sample. Statistical analysis of the HSP inhibitor Comet data was performed by calculation of median of each experimental point and then analyzing the differences between the means of the median values clustered by treatment. Apoptosis analysis Apoptosis induction was evaluated at single cell level through TUNEL assay on PBMCs samples taken before, 1?h and 24?h after the GXT. Lymphocytes were centrifuged, rinsed twice with PBS and fixed with paraformaldehyde (4% in PBS) at 4?��C for Phosphoprotein phosphatase 1?h. After incubation, cells were re-suspended in 500?��l PBS, gently dropped on a clean dry slide coated with poly-l-lysine (BD Biosciences) and air dried. After rinsed with PBS, cells were incubate in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) freshly prepared for 2?min on ice. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labeling (TUNEL) using ��In situ cell detection Kit, Fluorescein�� (Roche Applied Sciences, Germany). This assay is based on the identification of DNA strand breaks, generated during apoptosis, by labeling free 3��-OH termini with the addition of fluorescein dUTP at strand breaks by terminal deoxynucleotidyl transferase (TdT). Cells were labeled with fluorescein dUTP for 1hr at 37?��C in a humidified chamber in the dark. After, slides were washed 3 times with PBS and mounted in glycerol 50% PBS for the analysis under a fluorescence microscope (Olympus BX41). Apoptosis frequency was evaluated by scoring the number of TUNEL positive nuclei on at least 1000 cells analyzed. Mean and SEM were calculated from three separate cell samples, and all the experiments were performed in triplicate. Statistical analyses All statistical analyses were performed using IBM SPSS Statistics 18 (IBM Corporation). After testing whether data were normally distributed (Shapiro�CWilk test), an analysis of variance (ANOVA) with repeated measures for time (pre-training FTY720 molecular weight and 12 weeks) and group (trained and control) was performed. In the case of non-homogeneity of variances revealed by the Mauchly's sphericity test (p