After approximately one-year post SHIV challenge, the trispecific antibody treatment animals were injected intravenously with anti-CD8β mAb CD8b255R1 (National Institutes of Health Nonhuman Primate Reagent Resource Program) (50mg/kg). Whole blood was obtained and stained with fluorescently conjugated antibodies at different time points before and after infusion to monitor absolute counts of lymphocyte subsets in the blood using fluorescent bead containing trucount tubes (BD biosciences) as previously described (Pegu et al., 2015 (link)). In brief, the following antibodies were used to monitor the various lymphocyte subsets – anti-NHP CD45 FITC (clone D058-1283, BD Biosciences), anti-CD20 Pacific Blue (clone 2H7, BioLegend), anti-CD14 BV510 (M5E2, BioLegend), anti-CD69 BV605 (clone FN50, BioLegend), anti-CD16 BV711 (clone 3G8, BioLegend), anti-CD8 BV785 (clone RPA-T8, BioLegend), anti-CD4 Alexa Flour 700 (clone OKT4, BioLegend), anti-CD3 APC-Cy7 (clone SP34-2, BD Biosciences), anti-HLA-DR PE-Cy5.5 (clone TU36, ThermoFisher) and anti-CD159a PE-Cy7 (clone REA110, Milenyi Biotec). In addition, plasma viremia was quantified for up to 6 months post CD8+ T cell depletion.
Anti cd3 apc cy7 clone sp34 2
Manufactured by BD
Anti-CD3 APC-Cy7 (clone SP34-2) is a fluorochrome-conjugated monoclonal antibody that binds to the CD3 epsilon chain of the T cell receptor complex. It can be used in flow cytometry applications to identify and characterize T cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti hla dr pe cy5.5 clone tu36, by Thermo Fisher Scientific (1 mentions)
Anti cd95 pe cy5 clone dx2, by BD (1 mentions)
Anti pd 1 bv421 clone eh12.2h7, by BioLegend (1 mentions)
Facs ariaιι, by BD (1 mentions)
Anti cd4 fitc, by BioLegend (1 mentions)
Golgistop monensin, by BD (1 mentions)
Anti ifn γ pe clone 4s b3, by BioLegend (1 mentions)
Anti cd8 percp, by BioLegend (1 mentions)
Facsdiva, by FlowJo (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
3 protocols using anti cd3 apc cy7 clone sp34 2
1
CD8+ T Cell Depletion Monitoring
2
Characterization of CD4+ Tfh Cells
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Mononuclear cells isolated from LN were stained with anti-CD3-APC-Cy7 (clone SP34-2, 5 μL, cat #557757), anti-CD28-PE-CF594 (clone CD28.2, 5 μL, cat #562296), and anti-CD95-PECy5 (clone DX2, 10 μL, cat #559773) from BD Biosciences; anti-CD4-BV650 (clone OKT4, 2 μL, cat #317436), anti-PD-1-BV421 (clone EH12.2H7, 5 μL, cat # 3299220), and anti-CD200-PE (clone OX-104, 5 μL, cat #329206) from Biolegend; anti-CD8-FITC (clone 3B5, 5 μL, cat #MHCD0801-4) from Thermo Fisher Scientific; and Aqua Live/Dead Fixable Aqua from Invitrogen (AmCyan, 2 μL of 1:20 PBS dilution, cat. L34957). Sorting of CD4+ Tfh (PD1+CD200hi) was performed using a FACS AriaΙΙ (BD Biosciences) in samples collected before, during FTY720 treatment, at ART interruption, and 29 days after ART interruption. Post-sorting FACS analysis determined that sorted CD4+ T cell subsets were on average >96% pure.
3
HTLV-1 Tax-specific CD8+ T-cell Quantification
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HTLV-1 Tax-specific CD8+ T-cell frequencies were measured by flow cytometric analysis of gamma interferon (IFN-γ) induction after specific stimulation as described previously (44 (link)). Briefly, PBMCs were cocultured with autologous herpesvirus papio-immortalized B-lymphoblastoid cell lines (B-LCL) pulsed with peptide pools (at a final concentration of 2 μM for each peptide) using panels of overlapping peptides spanning HTLV-1 Tax amino acid sequences in the presence of GolgiStop (monensin; BD) for 6 h. Intracellular IFN-γ staining was performed using a Cytofix/Cytoperm kit (BD) and Live/Dead fixable aqua dead cell stain kit (Invitrogen), anti-CD3 APC-Cy7 (clone SP34-2; BD), anti-CD4 FITC, anti-CD8 PerCP, and anti-IFN-γ PE (clone 4S.B3; BioLegend). Stained cells were analyzed by BD FACSCanto II with FACSDiva v8.0.1 and FlowJo v9.2. Specific CD8+ T-cell frequencies were calculated by subtracting nonspecific IFN-γ+ CD8+ T-cell frequencies from those after peptide-specific stimulation. Specific CD8+ T-cell frequencies less than 0.02% of CD8+ T cells were considered negative.
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