T cell immunophenotyping and CD4+ and CD8+ T-cells relative count was performed on fresh blood samples anticoagulated with EDTA, using the antibodies PerCP anti-human CD3 (Clone: HIT3a), PE anti-human CD4 (Clone: RPA-T4), APC anti-human CD8 (Clone: RPA-T8); the samples were stained with Alexa 700 anti-human HLA-DR (Clone: L243) and FITC anti-human CD38 (Clone: HB-7) (All antibodies from BioLegend, San Diego CA, USA) to determine the immune activation. Attune NxT Flow Cytometer (Thermo Fisher Walthman, MA, USA) was used to acquire 10,000 events in the lymphocyte gate. The data was analyzed with Attune NxT Flow Cytometer Software version 2.6 (Thermo Fisher, Walthman, MA, USA). The normalization was determined by the proportion of the HLA-DR+, CD38+ as well as HLA-DR+ and CD38+ expression or MFI with the CD4+ and CD8+ T-cells relative count, respectively. The fold change analysis was calculated by the log2 fold difference of HLA-DR+, CD38+ and, HLA-DR+ and CD38+ expression and MFI in CD4+ and CD8+ T-cells of HIV+ groups divided by the expression or MFI of the same cell subpopulations in HIV- controls [18 (link), 19 (link)]. The CD4+/CD8+ ratio was calculated using the previously determined absolute CD4+ and CD8+ T-cells count. Serum levels of sCD14 and sCD163 were quantified by ELISA (both CUSABIO, Houston, TX, USA) according to the manufacturer instructions.
Apc anti human cd8
Manufactured by BioLegend
Sourced in United States
APC anti-human CD8 is a fluorochrome-conjugated monoclonal antibody that binds to the CD8 receptor expressed on the surface of T lymphocytes. It is used for the identification and enumeration of CD8-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Pe cy7 anti human cd4, by BioLegend (4 mentions)
Pe anti human cd4, by BioLegend (2 mentions)
Pe anti human cd3, by BioLegend (2 mentions)
Paci cblue anti human cd3, by BioLegend (2 mentions)
Ec800 cell analyzer, by Sony (2 mentions)
Trucount tube, by BD (2 mentions)
Pe anti human cd45, by BioLegend (2 mentions)
Fitc anti human cd38, by BioLegend (1 mentions)
Percp anti human cd3, by BioLegend (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Anti human cd3 fitc, by BD (1 mentions)
D biotin, by Thermo Fisher Scientific (1 mentions)
Pe anti human hla a2 antibody, by BioLegend (1 mentions)
Fetal bovine serum (fbs), by Lonsera (1 mentions)
Apc anti human cd69, by BioLegend (1 mentions)
Pe conjugated streptavidin, by BioLegend (1 mentions)
Anti human cd28 antibody, by BioLegend (1 mentions)
Percp anti human ifn γ, by BioLegend (1 mentions)
Fitc anti human granzyme b, by BioLegend (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Targetmol (1 mentions)
14 protocols using apc anti human cd8
1
T-Cell Immunophenotyping and Activation in HIV
2
Multiparameter Flow Cytometry of Activated T cells
3
Osteosarcoma T cell co-culture assay
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The pre-activated T cells were co-cultured with 143B and SJSA-1 osteosarcoma cells by using a transwell culture system with 0.4 μm pore polyester membrane. Briefly, the pre-activated T cells were seeded in 6-well plates at 1 × 106 cells/well, and osteosarcoma cells were seeded at 1 × 105 cells/well in Transwell chambers. After 48 h of co-culture, the suspensions CD8+ T cells were harvested and resuspended in staining buffer (DPBS containing 3% FBS) with the indicated antibodies: APC anti-human CD8 (Cat.344721, BioLegend), PE anti-human/mouse Granzyme B (Cat.372207, BioLegend), Brilliant Violet 605™ anti-human IFN-γ (Cat.502535, BioLegend), and analyzed by flow cytometry.
4
Comprehensive Immunologic Assay Protocol
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Antibody were purchased from BioLegend, including FITC anti-human HLA-A2 antibody (Cat#343303), PE anti-human HLA-A2 antibody (Cat#343305), anti-human CD28 antibodies (Cat#302901), APC anti-human-CD69 (Cat#310909), APC anti-human CD8 (Cat#344721), PerCP anti-human IFN-γ (Cat#502524), and FITC anti-human granzyme B (Cat#515403). Flex-T monomer (Cat#280003) and PE conjugated streptavidin (Cat#405203) were purchased from BioLegend. Ficoll-Paque PLUS (Cat#17144003) was from GE and DMSO (Cat#D2650) was from Sigma-Aldrich. IMDM (Cat#SH30228.01) and penicillin-streptomycin (Cat#SV30010) were from HyClone, and FBS (Cat#S711-001S) was purchased from LONSERA. PBS (Cat#C10010500BT) was from Gibco (Waltham, Massachusetts, USA). D-biotin (Cat#2110450) was purchased from Invitrogen (Waltham, Massachusetts, USA), and Mitomycin C (Cat#50-07-7) was from Sinochem Holdings (Beijing, China). CFSE (Cat#T6802) was from Targetmol (Boston, Massachusetts, USA). EasySep Human CD8+ T Cell Isolation Kit (Cat#17953) was from Stem Cell Technologies (Vancouver, British Columbia, Canada), and IL-2 (recombinant human interleukin-2(125Ala) injection) was from SL PHARM (Beijing, China). Leuko act cktl with GolgiPlug (Cat#550583) was purchased from BD.
5
Immune Cell Characterization with Fluorescent Markers
6
Multicolor Flow Cytometry Phenotyping
7
Multicolor Flow Cytometry of Hematopoietic Cells
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The blood cells collected in heparin anticoagulant tubes were washed with PBS and incubated with the antibody panel for 30–45 minutes at 4°C in the dark. Then, the cells were lysed with Pharm Lyse (Cat# 555899, BD Biosciences, San Jose, CA, USA) for 4 minutes. After centrifugation, the samples were suspended in PBS for data acquisition by flow cytometry (BD FACSCanto II, Beckman Coulter). Fluorochrome-conjugated monoclonal antibodies to the following human or mouse antigens were used: FITC-anti mouse CD45 (Biolegend, San Diego, CA, USA, Cat# 147710, RRID: AB_2563541), PE-anti human CD45 (Biolegend, Cat# 304039, RRID: AB_314395), FITC-anti human CD45 (Biolegend, Cat# 304038, RRID: AB_314393), PE-anti human CD3 (Biolegend, Cat# 300308, RRID: AB_314043), APC-anti human CD19 (Biolegend, Cat# 392506, RRID:AB_2750096), PE-Cy7 anti human CD4 (Biolegend, Cat# 357410, RRID: AB_2565661), APC-anti human CD8 (Biolegend, Cat# 344722, RRID: AB_2075390). A FACSCalibur instrument and Cellquest software were used for flow cytometry analysis.
8
Tumor Immune Profiling by Flow Cytometry
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Tumour samples were cut into small pieces and digested in RPMI-1640 supplemented with collagenase at 1 mg/mL (#C1889 Sigma USA). Digestion lasted for 1 hour at room temperature and filtered through a 70 µm filter. Single-cell suspensions were surface stained in fluorescence activated cell sorter (FACS) buffer (phosphate buffer solution (PBS), supplemented with 1% fetal bovine serum (FBS)) with the following antibodies for flow cytometric analysis, including FITC Zombie Green (#423 111 Biolegend USA), PE Anti-human CD3 (#344 806 Biolegend USA), APC Anti-human CD8 (#344 721 Biolegend USA), Brilliant Violet 421 Anti-human CD39 (#328 214 Biolegend USA), PE-Cy7 Anti-human PD-1 (#561 272 BD USA). Flow data were analysed by FlowJo V.10.0.
9
Phenotypic Profiling of PBMCs
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Lymphoprep® (Axis-Shield PoC AS, Oslo, Norway) was used to separate the whole blood into peripheral blood mononuclear cells (PBMCs). PBMCs were stained with antibodies, including FITC anti-human TCRαβ (BD Biosciences Pharmingen, San Diego, CA, USA), PE/Cy7 anti-human CD3 (Biolegend, San Diego, CA, USA), Paci cBlue anti-human CD3 (Biolegend), PE anti-human CD4 (Biolegend), PE/Cy7 antihuman CD4 (Biolegend), PE anti-human CD8 (Biolegend), APC anti-human CD8 (Biolegend), PE antihuman CD45RO (BD Biosciences Pharmingen), PE anti-human CCR6 (BD Biosciences Pharmingen), APC anti-human CCR4 (Biolegend), and APC anti-human CXCR3 (Biolegend), in a dark place at 4℃ for 30 min.
The PBMCs were xed in intracellular staining of RORγt and intracellularly stained using Fixation/Permeabilization Concentrate (eBioscience, San Diego, CA, USA), Fixation/Permeabilization Diluent (eBioscience), and Permeabilization buffer (eBioscience) with PE anti-human RORγt antibody (BD Biosciences Pharmingen) in a dark place at 4℃ for 45 min. The Cell Analyzer EC800 (Sony Biotechnology, Tokyo, Japan) was used for ow cytometric analysis. The data analysis was performed using FlowJo 7.6.5 software (TreeStar Inc, Ashland, OR, United States).
The PBMCs were xed in intracellular staining of RORγt and intracellularly stained using Fixation/Permeabilization Concentrate (eBioscience, San Diego, CA, USA), Fixation/Permeabilization Diluent (eBioscience), and Permeabilization buffer (eBioscience) with PE anti-human RORγt antibody (BD Biosciences Pharmingen) in a dark place at 4℃ for 45 min. The Cell Analyzer EC800 (Sony Biotechnology, Tokyo, Japan) was used for ow cytometric analysis. The data analysis was performed using FlowJo 7.6.5 software (TreeStar Inc, Ashland, OR, United States).
10
Evaluation of Cytokine Production in PBMCs
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Evaluation of capacity to produce IL-17A and IL-10 was performed. PBMCs maintained in RPMI1640 were stimulated with 50 ng/ml of phorbol-myristate acetate (PMA) (Sigma-Aldrich, St Louis, MO, USA) and 750 ng/ml of ionomycin (Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences Pharmingen) at 37℃ under 5% CO 2 for 4 h. PBMCs were stained with antibodies, including FITC anti-TCRαβ (BD Biosciences Pharmingen), PE/Cy7 anti-human CD4 (Biolegend), APC anti-human CD8 (Biolegend), and Paci cBlue anti-human CD3 (Biolegend), in a dark place at 4℃ for 30 min. PBMCs were xed and intracellularly stained using BD Cyto x and Perm/Wash (BD Biosciences Pharmingen) with PE anti-human IL-17A antibody (eBioscience) and PE anti-human IL-10 antibody (Biolegend) in a dark place at 4℃ for 30 min. Cell Analyzer EC800 (Sony Biotechnology) was used for ow cytometric analysis. FlowJo 7.6.5 software (TreeStar Inc, Ashland, OR, United States) was used for data analysis.
All studies were approved by the Ethics Committee of the Yokohama City University Graduate School of Medicine (number: B160804004). Informed consent was obtained from cases, parents and/or their guardians, and healthy adults for normal control.
All studies were approved by the Ethics Committee of the Yokohama City University Graduate School of Medicine (number: B160804004). Informed consent was obtained from cases, parents and/or their guardians, and healthy adults for normal control.
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