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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).

To measure IL-9 and IL-33 expressing T cells, Rhesus PBMCs were stimulated with 50 ng/ml of PMA (cat # 1201/1, Fisher Scientific) and 2.5 μM of Ionomycin in DMEM medium and incubated for 4 h in the presence of GolgiStop (BD-Biosciences) at 37°C. Cells were washed at 350 g for 5 min with MACS buffer (MACS buffer+1% BSA). Cells were stained with live dead stain (cat # L34961, Molecular Probes) and incubated for 20 mins at room temperature. Cells were washed and stained with cell surface markers (CD4-BB790 (BD-custom), CD19-AF700(Beckman Coulter- cat # A78837), CD20-APC-Cy7(BD Biosciences cat # 335794), IgM-BV605 (BD-Biosciences- cat # 562977, CD3-BV650 (BD Biosciences cat # 563916), CD8-BUV496-cat # 564805), incubated for 1 hr. at 4°C. Cells were washed twice with MACS buffer and fixed/permeabilized according to manufacturer’s protocol (BD-Cytofix/Cytoperm Fixation/Permeabilization kit- Cat # 554714). After fixation/permeabilization cells were stained with IL-9-AF647 (BD Biosciences cat # 560813), incubated for 30 min at 4°C, washed twice in BD Perm/Wash buffer and analyzed by flow cytometry.

Plasmablasts were isolated from freshly isolated peripheral blood mononuclear cells (PBMCs) collected seven days after Sanaria PfSPZ Vaccine immunization as previously described (Kisalu et al., 2018 (link)). Briefly, PBMCs were stained for viability with Aqua LIVE/DEAD (Thermo Fisher Scientific) followed by surface staining of the following markers: CD3-PE/Cy7 (BD Bioscience), CD19-FITC (BD Bioscience), CD20-APC/Cy7 (BD Bioscience), CD27-APC (Thermo Fisher Scientific), and CD38-PE (BD Bioscience). Plasmablasts were gated as live CD3⁻CD20⁻CD19⁺CD27⁺CD38⁺ and single cell sorted using a BD FACS Aria II (BD Immunocytometry Systems) into 96-well PCR plates containing 20 μL/well of RT reaction buffer from the SuperScript^™ First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific). Plates were snap frozen on dry ice and stored at −80°C.