The different groups of cells were co‐cultured at 37°C for 4 h and after brief centrifugation, the CD8+ T cells were isolated. The isolated T cells were stained with APC‐anti‐CD8 (BD Biosciences) alone or APC‐anti‐CD8 and PE‐anti‐IFN‐γ (BD Biosciences) or BV421‐anti‐granzyme B (Abcam). The cells were analyzed by flow cytometry for the purity and their function on the CytoFlex analytic flow cytometer (Beckman‐Coulter), and the data were analyzed by Flowjo software (Tree Star). In addition, the adherent tumor cells were stained with antibodies, followed by immunofluorescence.
Anti cd8 apc
Manufactured by BD
Sourced in United States
Anti-CD8-APC is a monoclonal antibody conjugated to the fluorescent dye allophycocyanin (APC). It is designed to detect and quantify CD8-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 fitc, by BD (14 mentions)
Anti cd4 pe, by BD (9 mentions)
Anti ifn γ pe, by BD (8 mentions)
Anti cd4 pe cy7, by BD (7 mentions)
Anti cd4 fitc, by BD (7 mentions)
Anti cd4 percp, by BD (5 mentions)
Anti cd4 apc, by BD (5 mentions)
Anti cd4 pacific blue, by BD (5 mentions)
Anti cd3 pe, by BD (5 mentions)
Facscalibur, by BD (4 mentions)
Anti hla dr pe, by BD (4 mentions)
Golgiplug, by BD (4 mentions)
Anti cd4 v450, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Facsaria 2, by BD (4 mentions)
Aim 5, by Thermo Fisher Scientific (4 mentions)
Anti cd69 pe, by BD (4 mentions)
Anti foxp3 pe, by BD (4 mentions)
Anti cd4 apc cy7, by BD (3 mentions)
Anti ifn γ pe cy7, by BD (3 mentions)
71 protocols using anti cd8 apc
1
Cytometry Analysis of CD8+ T Cells
2
Peptide-Stimulation Assay for T-Cell Response
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In vitro cultured PBMC's were harvested, washed, and aliquoted at 2–4 × 105 cells/well in a round bottom 96 well-plate. The in vitro cultured PBMC's were stimulated for 4 h at 37°C, 5% CO2 with, for the matrix analysis, each of the 15 column and 15 row mixes (1 μM/peptide), and for the epitope identification with a single peptide (0.8 μM). After 1 h of stimulation 1 μg/mL Brefeldin A (BD Biosciences) was added. The cells were subsequently permeabilized (Becton Dickinson Permeabilizing solution 2) and stained with anti-CD3-Pacific Blue, anti-CD4-PerCp, anti-CD8-APC, anti-CD69-PE, and anti-IFNγ-FITC, according to the “FastImmune” protocol (Becton Dickinson). The cells were subsequently analyzed by flow cytometry using a LSRII (BD Biosciences). The gating strategy is illustrated in Supplementary Figure S4 . Staining of more than 0.8% of CD8+ or CD4+ T cells was considered positive.
3
Multicolor Flow Cytometry for Lymphoid Populations
4
Characterization of NPM1-mutant CTL Phenotype
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NPM1mut-specific CTL products were characterized for phenotype by monoclonal antibody staining and flow cytometry. Anti-CD3 FITC, anti-CD4 PE, anti-HLA-DR PE, anti-CD8 APC, CDγδ FITC, anti-CD56 Pc5.5, anti-CD14 FITC, anti-CD56 PE, anti-CD3 Pc5.5, anti-CD19+ CD20 APC, anti-CCR7 FITC, and anti-CD45RA PE (Becton Dickinson, Franklin Lakes, NJ, USA) were employed.
5
Tetramer-Based Quantification of Antigen-Specific CTLs
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Peptide MRP3 765 -specific tetramer was used for the detection of peptide Mizukoshi et al. Phase I Trial of MRP3-Derived peptide for HCC. vaccine-induced CTLs. Peptide HIVenv 584 -specific tetramer was used as a negative control of tetramer assay. All tetramers were purchased from Medical Biological Laboratories Co., Ltd. (Nagoya, Japan). PBMCs were stained with anti-CD8-APC (Becton Dickinson, Tokyo, Japan), anti-CCR7-FITC (eBioscience, Tokyo, Japan), anti-CD45RA-PerCP-Cy5.5 antibodies (eBioscience, Tokyo, Japan), and with tetramer-PE for 30 min at room temperature. Cells were washed, fixed with 0.5% paraformaldehyde/PBS, and analyzed using a Becton Dickinson FACSAria II system. In the tetramer assays with the negative control tetramer, we did not observe more than 0.03% tetramer-positive cells in any assay. Based on the results of the negative control, responses to MRP 765 -specific tetramer were considered positive if more than 0.03% tetramer positive cells were detected. At least 1,000,000 PBMCs were acquired for each tetramer assay. For the detection of Tregs and MDSCs, the following anti-human monoclonal antibodies were used: anti-CD4 (Becton Dickinson), anti-CD14 (Becton Dickinson), anti-CD25 (Becton Dickinson), anti-CD127 (Becton Dickinson), and anti-HLA-DR (Becton Dickinson).
6
Flow Cytometry Analysis of Immune Cell Phenotypes
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The fluorochrome-labeled monoclonal antibodies (mAbs) specific for human monocyte surface and intracellular molecules were used for flow cytometry analysis. The mAbs specific for T cell surface molecules anti-CD14-BV510 (Clone-MφP9), anti-CD3-Alexa-fluor700 (Clone-UCHT1), anti-CD4-PECy7 (Clone-SK3), anti-CD4-V450 (Clone-RPA-T4), anti-CD8-APC (Clone-RPA-T8), anti-TNF-α-PE (Clone-MAb11), anti-IL-6-FITC (Clone-AS12), anti-IL-12-V450 (Clone-C11.5), anti-CD80-FITC (Clone-L307.4), anti-CD81-FITC (Clone-JS-81), Cytofix/Cytoperm and GolgiPlug were obtained from BD Biosciences (San Jose, CA). Anti-CD8-APC-Fluor-780 (Clone-RPA-T8) was purchased from eBioscience (San Diego, CA). Anti-CD40-PercpCy5.5 was purchased from BioLegend (San Diego, CA). Anti-pig SLA-class-I-FITC (Clone-JM1E3) and anti-human CD9-FITC (Clone-MM2/57) were obtained from Bio-Rad (Hercules, CA). Anti-SLA class-II-DR-FITC (clone 2E9/13) was obtained from LifeSpan BioSciences (Seattle, WA). Fetal bovine serum (FBS), Dulbecco's modified eagle medium (DMEM), and RPMI-1640 medium were purchased from Thermo-Fisher Scientific (Waltham, MA). CD14-microbeads, CD4 cell purification kits, and pan-T-cell purification kits were obtained from Miltenyibiotic (Cambridge, MA). Recombinant human tumor necrosis factor alpha (hTNF-α) and interferon gamma (hIFN-γ), and porcine IFN-γ (pIFN-γ) were purchased from R&D System (Minneapolis, MN).
7
Mouse and Human gp100 Peptide Protocol
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Mouse gp10025–33 (mgp100, EGSRNQDWL) and human gp10025–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea). CD8 microbeads were purchased from Miltenyi Biotec (Auburn, AL, USA). All antibodies used for flow cytometry were purchased from BD Bioscience, including anti-Thy1.1-FITC, anti-CD3-FITC, anti-B220-FITC, anti-CD62L-FITC, anti-Ly-6C-FITC, anti-CD19-PE, anti-CD8-PE, anti-CD44-PE, anti-CD8-PE-Cy5, anti-Thy1.1-PE-Cy5, anti-CD11b-PE-Cy5, anti-CD4-APC, anti-CD8-APC, and anti-CD45-APC. Recombinant human IL-2 (Proleukin) was purchased from Novartis, and the Fc fusion protein of human IL-7 (IL7-Fc), which consist of the extracellular domain of human IL-7 (aa 26–177) fused to the N-terminus of the Fc portion of a mutant human IgG1, was obtained from AdipoGen (Seoul, Korea). The CellTrace CFSE Cell Proliferation Kit was purchased from Invitrogen (Carlsbad, CA, USA).
8
Flow Cytometric Analysis of T-Cells
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EDTA-treated whole blood was incubated using the corresponding monoclonal antibodies: anti-CCR7-FITC, anti-CD57-PE, anti-CD3-PerCP5.5, anti-CD45RA-PCy7 and anti-CD8-APC (all from BDBiosciences, New York, NY, USA); anti-CD4-APC-AlexaFluor750 and anti-HLA-DR-PB (all from Beckman Coulter, Miami, FL, USA). Proportions of T-cells were analyzed by flow cytometry using a Navios Cytometer and Kaluza Software (Beckman Coulter, Miami, FL, USA) [40 (link)].
9
Cytotoxic T-cell Degranulation Assay
10
Comprehensive Immune Cell Profiling
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PBMCs, LNs, and tumor cell suspensions at 0.5x106 cells/sample were washed in PBS containing 2% FCS and 0.05% NaN3 (FACS-buffer). To investigate lymphocyte subtypes and their functions, staining was performed with fluorophore-conjugated antibodies: Blue Live/Dead stain, anti-CD4 Pacific-Blue, anti-CD8 APC, anti-CD19 APC-Cy7, and anti-CD56 PE (Becton Dickinson-BD, Franklin Lakes, NJ, USA). Cells were investigated with LSRFORTESSA (BD) and analyzed by using the FACS DIVA software (BD).
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