Computational protocol: Mobilization of Peripheral Blood Stem Cells and Changes in the Concentration of Plasma Factors Influencing their Movement in Patients with Panic Disorder

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Protocol publication

[…] Peripheral blood (PB) samples were lysed twice using BD Pharm Lyse lysing buffer (BD Bioscience) at room temperature for 10 min and subsequently washed in phosphate-buffered saline (PBS) with 2 % fetal bovine serum (FBS; Sigma) to yield total nucleated cells (TNCs). TNCs were stained for hematopoietic lineage markers using the following fluorescein isothiocyanate (FITC)-conjugated antibodies (Abs) against human proteins: CD2 (clone RPA-2.10); CD3 (clone UCHT1); CD14 (clone M5E2); CD16 (clone 3G8); CD19 (clone HIB19); CD24 (clone ML5); CD56 (clone NCAM16.2); CD66b (clone G10F5); and CD235a (clone GA-R2) (all from BD Bioscience). The cells were simultaneously stained for the panleukocytic marker, CD45 phycoerythrin (PE)-conjugated Abs, (clone HI30; BD Biosciences) and one of the following antigens CD 34-allophycocyanin (APC)-conjugated Abs, (clone 581; BD Bioscience) or CD133 (CD133/1, APC-conjugated Abs; Miltenyi Biotec). Additionally following FITC-conjugated isotype controls: Mouse IgG1, (clone MOPC-21), Mouse IgG2a, (clone G155–178), Mouse IgG2b, (clone 27–35), PE-conjugated isotype control Mouse IgG1, (clone MOPC-21) and APC-conjugated isotype control Mouse IgG1, (clone MOPC-21) (all from BD Bioscience) were used. In addition, APC-conjugated isotype control Mouse IgG1 (clone IS5-21F5; Miltenyi Biotec) were used. Staining was performed in PBS with 2 % FBS on ice for 30 min. Cells were subsequently washed, resuspended, and analyzed using an NAVIOS Flow Cytometer (Beckman Coulter). At least 106 events were acquired from each sample. The absolute numbers of VSEL and the absolute number of white blood cells were calculated (individually for each patient) per 1 ml PB based on the percent content of these cells as detected by flow cytometry. Kaluza software (Beckman Coulter) was used for analysis [, ]. [...] Distribution of continuous variables was examined with the Shapiro-Wilk test. Comparison of quantitative variables was performed with the Mann-Whitney-U test. Relative continuous variables were compared with the Wilcoxon test. Dichotomous variables comparison was performed with the use of the exact Fisher test. As the measure of statistical significance we used p < 0.05. All statistical analyses were performed using Statistica 10, by StatSoft, 2013. […]

Pipeline specifications

Software tools Kaluza, Statistica
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens