Tumor-specific CD4 T clones were stained systematically in PBS, 0.2% bovine serum albumin (BSA), 5 mM EDTA, and 0.2% NaN3 with either (i) FITC anti-CD3 (BioLegend), BV605 anti-CD4 (BioLegend), ECD anti-CD8 (BD), PE-Cy7 anti-SLAMF7 (BioLegend), PerCP-Cy5.5 anti-CD57 (BioLegend), APC (allophycocyanin) anti-CD28 (BioLegend), BV711 anti–PD-1 (BioLegend), PE anti-TCRαβ (BioLegend), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) or (ii) FITC anti-CD3 (BioLegend), BV605 anti-CD4 (BioLegend), APC anti-TRAIL (BioLegend), PE anti-FasL (BioLegend), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) at 4°C for 30 min. Data were acquired on LSR II (BD Biosciences) and analyzed using FlowJo software (v.10.4.2).
Anti cd3 fitc
Manufactured by BioLegend
Sourced in United States, United Kingdom, China
Anti-CD3-FITC is a monoclonal antibody conjugated with the fluorescent dye fluorescein isothiocyanate (FITC). It is designed to specifically bind to the CD3 receptor expressed on the surface of T cells, which plays a crucial role in T cell activation and signaling.
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Lab products found in correlation
Anti cd4 apc, by BioLegend (19 mentions)
Anti cd8 apc, by BioLegend (16 mentions)
Anti foxp3 pe, by BioLegend (8 mentions)
Anti cd8 pe, by BioLegend (8 mentions)
Anti cd56 apc, by BioLegend (7 mentions)
Anti cd4 pe cy7, by BioLegend (7 mentions)
Anti cd11b pe, by BioLegend (6 mentions)
Anti cd56 pe, by BioLegend (6 mentions)
Facscanto 2, by BD (6 mentions)
Anti cd8 apc cy7, by BioLegend (5 mentions)
Anti cd4 percp cy5, by BioLegend (5 mentions)
Anti cd4 fitc, by BioLegend (5 mentions)
Fc500, by Beckman Coulter (4 mentions)
Anti ifn γ apc, by BioLegend (4 mentions)
Anti cd4 bv605, by BioLegend (4 mentions)
Anti cd4 percp, by BioLegend (4 mentions)
Anti cd19 apc, by BioLegend (4 mentions)
Anti cd45 pe, by BioLegend (4 mentions)
Anti cd4 apc cy7, by BioLegend (4 mentions)
Anti cd25 pe, by BioLegend (4 mentions)
114 protocols using anti cd3 fitc
1
Comprehensive Phenotypic Analysis of Tumor-Specific CD4 T Cells
2
Influenza B Antibody Assay Using Flow Cytometry
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Sera samples for analysis were firstly treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) at 37 °C for 18 h followed by the inactivation of the RDE via incubation at 56 °C for 30 min.
Wells of a 96-well ELISA plate were coated with purified influenza B/Brisbane/60/2008 and B/Florida/4/2006 HA protein (400 ng/well) overnight at 4 °C in PBS. The plates were then washed twice with PBS and 100 µL of sera treated with RDE at 37 °C for 30 min. Plates were then washed five times with PBS and 106 freshly isolated murine PBMCs were added to each well. PBMCs isolated from NC group mice using Ficoll-Paque (GE Healthcare Life Sciences, Uppsala, Sweden ) were washed and resuspended in RF10 media (RPMI 1640, supplemented with 10% FCS, penicillin, streptomycin and l-glutamine; Life Technologies, Grand Island, NY, USA) and were then added to the washed wells and incubated for 5 h at 37 °C with 5% CO2. Finally, anti-mouse antibody FITC-antiCD3, PE-antiCD49b and BV421-antiCD107a (Biolegend; used at a 1:400 dilution) were added and incubated at 37 °C for 30 min in the dark. Cells were then fixed with 1% formaldehyde at 37 °C for 10 min and acquisition was performed on the LSRFortessaX-20 flow cytometer (BD Biosciences) with up to106 lymphocyte events collected. Samples were analyzed using FlowJo version 9.2 (Tree Star, Ashland, OR, USA).
Wells of a 96-well ELISA plate were coated with purified influenza B/Brisbane/60/2008 and B/Florida/4/2006 HA protein (400 ng/well) overnight at 4 °C in PBS. The plates were then washed twice with PBS and 100 µL of sera treated with RDE at 37 °C for 30 min. Plates were then washed five times with PBS and 106 freshly isolated murine PBMCs were added to each well. PBMCs isolated from NC group mice using Ficoll-Paque (GE Healthcare Life Sciences, Uppsala, Sweden ) were washed and resuspended in RF10 media (RPMI 1640, supplemented with 10% FCS, penicillin, streptomycin and l-glutamine; Life Technologies, Grand Island, NY, USA) and were then added to the washed wells and incubated for 5 h at 37 °C with 5% CO2. Finally, anti-mouse antibody FITC-antiCD3, PE-antiCD49b and BV421-antiCD107a (Biolegend; used at a 1:400 dilution) were added and incubated at 37 °C for 30 min in the dark. Cells were then fixed with 1% formaldehyde at 37 °C for 10 min and acquisition was performed on the LSRFortessaX-20 flow cytometer (BD Biosciences) with up to106 lymphocyte events collected. Samples were analyzed using FlowJo version 9.2 (Tree Star, Ashland, OR, USA).
3
Multiparametric Flow Cytometry Analysis
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Cultured MNCs were collected from each well and centrifuged. The cell pellet was resuspended in PBS. The sample to be tested was incubated with the following antibodies at 4 °C for 30 min. CD45+ lymphocytes, CD3+ T cells, CD3+ CD4+ T cells, and CD3+ CD8+ T cells were detected with the FITC anti-CD3/PE anti-CD8/PerCP anti-CD45/APC anti-CD4 detection kit (ACEA Biosciences, China). CD3− CD56+ CD16+ NK cells and CD3− CD19+ B cells were detected using a FITC anti-CD3/PE anti-CD16+ CD56/PerCP anti-CD45/APC anti-CD19 detection kit (ACEA biosciences). CD4+ CD25+ Foxp3+ Treg cells were detected with FITC anti-human-CD25 (BioLegend), PerCP/Cy5.5 anti-human-CD4 (BioLegend), and PE anti-human-Foxp3 (BioLegend). CD38+ CD3− CD56+ NK cells were detected with PE anti-CD38 (BioLegend), FITC anti-CD3 (BioLegend), and APC anti-CD56 (BioLegend). Lymphocyte subtypes within MNCs were measured with flow cytometry.
4
Flow Cytometry Immunophenotyping Protocol
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The human monoclonal antibodies (mAbs) used for flow cytometry in this article included FITC-anti-CD3, FITC-anti-CD80, FITC-anti-CD163, FITC-anti-CD8α, FITC-anti-CD14, PE-anti-CD40, APC-anti-IFN-γ (all from BioLegend), BB515-anti-TLR2, and PE-anti-CD206 (all from BD Biosciences). Cells were scrapped from culture plates and incubated with human AB serum to saturate non-specific mAb binding before staining with the indicated fluorophore-labeled mAbs. For intracellular IFN-γ staining, cells were stimulated with Cell Activation Cocktail (with Brefeldin A; BioLegend) for 6 h according to the manufacturer's protocol. All data were collected using Novocyte flow cytometer (ACEA) and analyzed with FlowJo software.
5
Cytometry and Neutralization Assay for Mycobacterial Antigens
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For flow cytometry, we used FITC anti-CD3, PE anti-CD27, PE/Cy7 anti-NKp46, FITC anti-CD8, PE anti-CD11b, PE anti-CD56, allophycocyanin KLRG1, (all from BioLegend). For neutralization, we used mAb to IL-4, IL-7, IL-17, IL-21, or isotype-matched control antibody (eBioscience). Recombinant mouse IL-21 was obtained from eBioscience. We used γ-irradiated M. tb H37Rv (γ-M. tb), ESAT6, and Ag85a (all from BEI Resources), and the BCG Tice strain (Organon USA Inc.).
6
Multiparameter Flow Cytometry of T Cell Activation
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CD4+ T cells were surface stained for 30 min using the following murine monoclonal antibodies: APC-anti 4-1BB (Cat. 309810), PE-anti Gal-3 (Cat. 126706), FITC-anti CD3 (BioLegend) and LIVE/DEAD (Life Technologies). Intracellularly staining were preceded by blocking with 50 µg/ml mouse IgG for 15 min before staining with an anti-TNFα antibody (Franklin Lakes, USA). Cells were washed, fixed and analyzed on a NovoCyte Quanteon™ or Amnis® ImageStream® Imagine Flow Cytometer. Spectral overlap was compensated using antibody-coated beads (eBioscience). Gating was done on live cells using fluorescence minus one (FMO). Data were analyzed using IDEAS for windows version 6.2 and/or FlowJo for Mac software version 10.1.
Imagestream gating included cells in focus, single cells and CD3 positive cells. A membrane mask was defined based on the CD3 stain using morphology mask and erode. Membrane mask included the outer three pixels. Within this mask the aggregation of 4-1BB was calculated and the surface specific MFI values determined.
Imagestream gating included cells in focus, single cells and CD3 positive cells. A membrane mask was defined based on the CD3 stain using morphology mask and erode. Membrane mask included the outer three pixels. Within this mask the aggregation of 4-1BB was calculated and the surface specific MFI values determined.
7
Flow Cytometry for Immune Cell Quantification
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The flow cytometry procedure for identification and quantification of circulating inflammatory and immune cells was based on our previous report [34 (link)]. Prior to sacrificing the animals, peripheral blood mononuclear cells (PBMCs) were obtained from the tail vein using a 27# needle. PBMCs (1.0 × 106 cells) were triple-stained with FITC-anti-CD3 (BioLegend), PE-anti-CD8a (BD Bioscience, San Jose, CA, USA), and PE-Cy™5 anti-CD4 (BD Bioscience, San Jose, CA, USA). To identify CD4+CD25+Foxp3+ regulatory T cells (Tregs), PBMCs were triple-stained with Alexa Fluor® 488-anti-CD25 (BioLegend, San Diego, CA, USA), PE-anti-Foxp3 (BioLegend, San Diego, CA, USA), and PE-Cy™5 anti-CD4 (BD bioscience, San Jose, CA, USA) according to the manufacturer's protocol for the Foxp3 Fix/Perm buffer set. The numbers of CD3+CD4+ helper T cells, CD3+CD8+ cytotoxic T cells and CD4+CD25+Foxp3+ Tregs were analyzed using flow cytometry (FC500, Beckman Coulter, Brea, CA, USA).
Additionally, the numbers of inflammatory cells in circulation [i.e., CD11b/c, LyG6, vascular cell adhesion molecule (VCAM)-1], in ascites [macrophage migratory inhibitor factor (MIF), CD14, CD11b/c, LyG6, CD68/CD80, CD68/CD163], and in ABL (CD11b/c, MIF, Ly6G) were assessed using the flow cytometric method.
Additionally, the numbers of inflammatory cells in circulation [i.e., CD11b/c, LyG6, vascular cell adhesion molecule (VCAM)-1], in ascites [macrophage migratory inhibitor factor (MIF), CD14, CD11b/c, LyG6, CD68/CD80, CD68/CD163], and in ABL (CD11b/c, MIF, Ly6G) were assessed using the flow cytometric method.
8
Identification and Quantification of Immune Cells
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The procedure and protocol of flow cytometry for identification and quantification of circulating and splenic immune cells were based on our previous report [44 (link)]. Briefly, the peripheral blood mononuclear cells (PBMCs) and splenocytes were obtained from the tail vein using a 27# needle. PBMCs and splenocytes (1.0 × 106 cells) were triple-stained with FITC-anti-CD3 (BioLegend), PE-anti-CD8a (BD Bioscience), and PE-Cy™5 anti-CD4 (BD bioscience). The numbers of CD3+CD4+ helper T cells and CD3+CD8+ cytotoxic T cells were analyzed using flow cytometry (FC500, Beckman Coulter).
9
Quantification of CMV-specific T-cells
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After 3 days of incubation in the presence or absence of vitamin C, 1 × 106 cells per sample were stimulated with peptide pool CMV pp65pp for one hour followed by addition of Brefeldin A (BioLegend) and incubation for another 15 h at 37 °C and 5% CO2. Cells were resuspended, washed and extracellularly stained using FITC anti-CD3, APC/Cy7 anti-CD8 and PerCP anti-CD4 (all BioLegend). Subsequently, cells were fixed, permeabilized and intracellularly stained with APC anti-TNF-α (BioLegend) using IntraPrep kit (purchased by Beckman Coulter) following the manufacturer’s instructions. Samples were acquired on a FACS Canto 10c, and gates were set based on the forward scatter versus side scatter properties of lymphocytes. At least 30,000 events were acquired in the CD3+ gate. Data were analyzed using FlowJo_v10.7.1 software.
10
Multiparametric Phenotyping of Antigen-Specific CD4+ T Cells
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Enriched CD4 T cells were purified by positive selection using MACS isolation microbeads (Miltenyi) and stained directly ex vivo using a combination of fluorescent pMHCII multimers, as described above and FITC anti-CD3 (BioLegend), BV605 anti-CD4 (BioLegend), PE-Cy7 anti-SLAMF7 (BioLegend), BV711 anti–PD-1 (BioLegend), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Irrelevant multimers were used as negative controls. Data were acquired on LSR II (BD Biosciences) and analyzed using FlowJo software (v.10.4.2).
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