Lsr 2 multicolor flow cytometer

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.

Analysis of circulating immune cell phenotypes was performed both prior to transplant and at regular intervals following transplantation. Cell frequencies from flow cytometric analysis were combined with complete blood counts to calculate total numbers of circulating T cells and various T cell subsets. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation (BD Biosciences, Franklin Lakes, NJ) within 6 hours of phlebotomy. PBMCs (1.5×10^6) were then incubated with antibody mixtures at the appropriate titer for 15 minutes and washed twice. For assessment of intracellular markers, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s direction following surface staining. Flow cytometric data was acquired immediately using a BD LSR II multicolor flow cytometer (BD Biosciences). All flow data was analyzed using FlowJo (Tree Star, San Carlos, CA).
Surface markers were stained with the following monoclonal antibodies (mAbs): CD3 PacBlue, CD3 APC-Cy7, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD127 PE-Cy7, PD-1 APC, LFA-1 APC, CD20 APC-Cy7 (all BD Biosciences), CD95 PacBlue, CD69 FITC (Invitrogen, Grand Island, NY), and CD25 PE (Miltenyi Biotech, San Diego, CA). Intracellular staining for FoxP3 was performed using FoxP3 Alexa488 (Biolegend, San Diego, CA).