Ficoll density gradient centrifugation

Flow cytometric analysis was performed up to 3 times pre-transplant and serially post-transplant to characterize peripheral blood immune cell phenotypes. Total T cells and T cell subsets were quantified by complete blood cell count and flow cytometry. Fresh PBMCs were isolated by Ficoll density gradient centrifugation (BD Biosciences, Franklin Lakes, NJ). PBMCs were stained with the following mAbs: CD3 PacBlue, CD95 V450, CD3 Alexa 700, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD25 PE-Cy7, IFNy PE-Cy7, CD28 APC, TNF APC, VLA-4 APC, CD11a PE, CD45RA FITC, CD40 FITC, CCR7 APC, CD20 APC (all BD Biosciences). PBMCs (1.5×10⁶) were incubated with appropriately titered antibodies for 15min at 20°C and washed twice. Samples were acquired immediately on a BD LSR II multicolor flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, San Carlos, CA). For the stimulation assay, 1.5×10⁶ PBMCs were cultured in RPMI 1640 (Corning cellgro, Manassas, VA) supplemented with 10% fetal bovine serum and stimulated with 10µM phorbol 12-myristate 13-acetate (PMA) and 200nM ionomycin (Sigma-Aldrich, St. Louis, MO), with 1ul/ml GolgiPlug protein transport inhibitor for 5 h, +/− IL-15 (10ng/mL). PBMCs were were processed with BD Cytofix/Cytoperm Plus kit (BD 555028) per the manufactures recommendation prior to data acquisition.

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood collected before and after transplantation by Ficoll density gradient centrifugation according to the manufacturer’s protocol (BD Biosciences). PBMCs were re-suspended in FACS buffer (PBS containing 1% fetal bovine serum), and at least 10⁵ PBMCs were surface-stained with mAbs in the darkness at room temperature. Intracellular staining for Ki67 and Bcl-2 was carried out based on the protocol provided by manufacturer (BD Biosciences). Briefly, surface-stained cells were permeabilized/fixed with Perm/Fixation buffer (BD Biosciences) for 45 minutes on ice. Cells were stained with mAb specific for Ki67 and Bcl-2 at 4°C for 30 minutes. Cells were resuspended in FACS buffer and analyzed using LSR II polychromatic flow cytometry. Data analysis was performed using FlowJo software (Tree Star, San Carlos, CA).