PE-conjugated HLA-A*0201 RMSAPSTGGV tetramer (H3.3K27M-tetramer) was produced by the National Institute of Allergy and Infectious Disease tetramer facility within the Emory University Vaccine Center (Atlanta, GA) using the peptide synthesized by A&A Laboratories. PE-conjugated HLA-A*0201/RMSAPSTGGV Dextramer was purchased from Immudex. Cells were stained with tetramer (10 µg/ml) or dextramer in PBS containing 1% BSA for 15 min at 4°C (for tetramer) or room temperature (for dextramer), followed by surface staining for various T cell markers at 4°C. Cells were then washed with PBS containing 0.1% BSA. For some experiments, T cells were stained with tetramer, followed by anti–human CD3 FITC (344803; BioLegend), CD4-PerCPCy.5.5 (317427; BioLegend), CD8 APC (344722; BioLegend), CD69 FITC (11-0699-42; eBioscience), or PD-1-PECy7 (561272; BD Biosciences) along with the suitable isotype control antibodies. Intracellular cytokine staining was performed using Fixation/Permeabilization Solution kit (54714; BD Biosciences) according to the manufacturer’s instructions. T cells were then stained with anti–human Granzyme-B-BV421 (563389; BD Biosciences). The cells were acquired using a Sony SH800 flow cytometer and analyzed using FlowJo software v.10.
Anti human cd3 fitc
Manufactured by BioLegend
Sourced in United States
Anti-human CD3-FITC is a fluorescently labeled antibody that binds to the CD3 protein, which is a part of the T-cell receptor complex. This product can be used to identify and analyze T cells in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd8 pe, by BD (4 mentions)
Pe anti mouse cd45, by BD (2 mentions)
Pe anti mouse cd8a, by BioLegend (2 mentions)
Anti mouse cd45.1 fitc, by BioLegend (2 mentions)
Anti mouse cd45.2 pe, by BioLegend (2 mentions)
Apc anti human cd33, by BioLegend (2 mentions)
Anti human cd45 pe, by BD (2 mentions)
Anti cd127, by BioLegend (2 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Pacific blue anti human cd4, by BioLegend (2 mentions)
Pe anti human cd56, by BioLegend (2 mentions)
Facscalibur, by BD (2 mentions)
Cd8 apc, by BioLegend (1 mentions)
Cd69 fitc, by Thermo Fisher Scientific (1 mentions)
Cd4 percp cy5, by BioLegend (1 mentions)
Pd 1 pe cy7, by BD (1 mentions)
Sh800 flow cytometer, by FlowJo (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Apc anti human cd69, by BioLegend (1 mentions)
Anti human cd14 apc, by Thermo Fisher Scientific (1 mentions)
23 protocols using anti human cd3 fitc
1
Quantification of Antigen-Specific T Cells via HLA Tetramers
2
Multiparameter Flow Cytometry Analysis
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Primary antibodies including anti-mouse CD8a-PE (BioLegend, 100707, 53–6.7, 1:100), anti-mouse CD45-PE (BD Pharmingen,561087, 30-F11, 1:100), anti-mouse CD45.1-FITC (BioLegend, 110705, A20, 1:100), anti-mouse CD45.2-PE (BioLegend, 109807,104, 1:100), anti-human CD45-PE (BD Pharmingen, 555483, HI30, 1:100), anti-human CD33-APC (Biolegend,366605, P67.6, 1:100), anti-human CD3-FITC (BioLegend, 300305, HIT3a, 1:100), anti-human CD8-PE (BD Pharmingen, 555367, RPA-T8, 1:100) antibodies were used. Cells were run on Calibur for analysis or FACSAria for sorting.
3
Multiparameter Flow Cytometry Staining
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Cells were washed in FACS medium (PBS containing 2% FBS), and stained with primary antibodies including anti-human CD14-APC (eBioscience, 61D3, 1:100), anti-human CD4-APC (eBioscience, RPA-T4, 1:100), anti-human CD3-FITC (BioLegend, HIT3a, 1:100), anti-human CD8-PE (BD Pharmingen, 555367, 1:100), anti-human CD69-APC (BioLegend, FN50, 1:100), anti-human CD86-PE (BioLegend, IT2.2, 1:100), anti-CD206-APC (eBioscience, 19.2, 1:100), anti-CD11c (Biolegend, Bu15, 1:100), anti-hLAIR1 (eBioscience, NKTA255, 1:100), anti-CD45 (Biolegend, 2D1, 1:100), anti-CD25 (Biolegend, BC96, 1:100), or anti-CD127 (Biolegend, A019D5, 1:100). Flow data were analyzed by Flowjo software. Propidium iodide (PI) staining was used to exclude dead cells in analysis.
4
Multi-Marker Flow Cytometry Profiling
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Anti-human PDPN–PE (clone NZ-1.3, 12-9381-42, 1:100), anti-human CD45–PE-Cy7 (clone HI30, 25-0459-42, 1:100), anti-human CD31–biotin (clone WM59, 13-0319-82, 1:100) and anti-human or mouse ACTA2–eFluor 660 (clone 1A4, 50-9760-82, 1:500) were purchased from eBioscience. Anti-human CD31–PerCP (clone WM59, 303132, 1:50), anti-human CD235a–PE-Cy7 (clone HI264, 349112, 1:100), anti-human CD3–FITC (clone UCHT1, 300440, 1:100), anti-human CD14–PE-Cy7 (clone MSE2, 301813, 1:100), anti-human CD19–APC/Fire 750 (clone HIB19, 302258, 1:100), anti-human CD54 (ICAM1)–BV421 (clone HA58, 353132, 1:100) and anti-human CD34–FITC (clone 581, 343504, 1:100) were purchased from BioLegend. Anti-human CD45–APC-Cy7 (clone 2D1, 560178, 1:100), anti-human CD4–BUV395 (clone SK3, 563550, 1:200), anti-human CD8–BUV805 (clone SK1, 612889, 1:400) and anti-human CD25–BUV563 (clone 2A3, 612918, 1:400) were purchased from BD Biosciences.
5
Multicolor Flow Cytometry Immunophenotyping
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The following antibodies were used in this study: anti-human CD3 FITC (BioLegend), anti-human CD8 PE (BioLegend), anti-human CCR7 PE (BioLegend), anti-human CD45RA FITC (BioLegend), mouse anti-human CD69 Alexa Fluor 488 (BioLegend), and anti-human B220 APC (antigen-presenting cell; BioLegend). Chimeric FL-BCR expression was detected using anti-human IgG1 PE antibody (SouthernBiotech) or synthetic biotinylated cyclopeptides (GeneCust) and streptavidin conjugated with FITC or PE (Thermo Fisher Scientific). The CAR molecules were detected using goat cross-absorbed anti-human IgG antibody conjugated with DyLight 650 (Thermo Fisher Scientific). The CD19-CAR (FMC63 clone) molecules were detected using biotinylated protein L (Thermo Fisher Scientific) and streptavidin conjugated with FITC (Thermo Fisher Scientific).
6
CD107α Upregulation Assay for T Cells
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T cells (1 × 105) were cultured in AB-RPMI at 1 : 1 ratio with T2 cells ± peptide in the presence of GolgiStop™ and GolgiPlug™ (BD). T cells were also cultured in the presence of mitogen (positive control). Changes in the surface expression of CD107α were determined through the addition of anti-human CD107α-phycoerthrin (Biolegend – clone H4A3) to each culture. After 5 hr the cells were washed and co-stained with anti-human CD3-FITC (Biolegend – clone UCHT1) and CD8-peridinin chlorophyll protein (Biolegend – clone HIT8a). The cells were analysed using an Accuri C6 (BD) and data analysis was performed using cflow software (BD).
7
Multiparameter Flow Cytometry of Hematopoietic Cells
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BM, spleen (SPL), and peripheral blood (PB) were obtained from mice. Single-cell suspensions were prepared from each tissue following standard procedures. Erythrocytes were hemolyzed using BD Pharm Lyse buffer (BD Biosciences). Leukocytes were stained with fluorochrome-conjugated human antibody/mouse antibody for 20 min at room temperature in FACS buffer. After washing the labeled cells in 1x PBS, the cells were re-suspended in FACS buffer. Fluorescent signals were detected with a FACSVerseTM flow cytometer, and data were analyzed using FlowJo software (BD Biosciences). The following human antibodies were used to measure human engrafted hematopoietic cells and mouse hematopoietic cells: anti-human CD45-APC and anti-human CD66b-FITC were purchased from Beckman Coulter. Anti-mouse CD45-FITC, anti-human CD33-FITC, anti-human CD14-PE, anti-human CD11b-PE, anti-human CD41-PE, anti-human CD235a-FITC, anti-human CD3-FITC, anti-human CD19-APC were purchased from BioLegend.
8
Quantifying T Cell Subsets in Mice
9
Cytokine and Chemokine Analysis in moDCs
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Supernatants of moDCs and TU-CM-moDCs were harvested four days after monocyte separation or 24 hours after the stimulation of moDCs. The concentration of IL-6, IL-10, TNFα cytokines, and chemokine IL-8 was measured and validated using OptEIA kits (BD Biosciences) following the manufacturer’s instructions.
To determine which T-lymphocyte populations are responsible for the cytokine production, the T cells were stimulated with 1 μg/ml ionomycin and 20 ng/ml phorbol-myristic acetate (PMA) for 4 hours. The vesicular transport was inhibited by BD GolgiStop™ protein transport inhibitor (BD Biosciences) after the activation. The cells were then labeled with anti-human CD3-FITC, anti-human CD8-PE or CD4-PerCP, and anti-human CD25-PE antibodies (all from BioLegend). The samples were then fixed and permeabilized by BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences) and labeled again with anti-human CD8-PE or CD4-PerCP as well as with anti-human IFNγ-APC (BD Biosciences), anti-human IL-4-PE (R&D Systems), anti-human IL-10-Alexa Fluor 488, anti-human IL-17-PE (BioLegend). T cell staining panel is shown inS1 Fig . Fluorescence intensities were measured by Novocyte2000R Flow Cytometer (Agilent (Acea) Biosciences Inc., USA), and data were analyzed by the FlowJo v X.0.7 software (Tree Star).
To determine which T-lymphocyte populations are responsible for the cytokine production, the T cells were stimulated with 1 μg/ml ionomycin and 20 ng/ml phorbol-myristic acetate (PMA) for 4 hours. The vesicular transport was inhibited by BD GolgiStop™ protein transport inhibitor (BD Biosciences) after the activation. The cells were then labeled with anti-human CD3-FITC, anti-human CD8-PE or CD4-PerCP, and anti-human CD25-PE antibodies (all from BioLegend). The samples were then fixed and permeabilized by BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences) and labeled again with anti-human CD8-PE or CD4-PerCP as well as with anti-human IFNγ-APC (BD Biosciences), anti-human IL-4-PE (R&D Systems), anti-human IL-10-Alexa Fluor 488, anti-human IL-17-PE (BioLegend). T cell staining panel is shown in
10
Chemokine Receptor Profiling and Activation
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Recombinant human CCL3 (MIP-1α ), CCL4 (MIP-1β ), CCL11 (Eotaxin), CCL1 (MCP-1), IL-13, GM-CSF, IL-4, and IFNγ were obtained from ProSpec Protein-Specialists (East Brunswick, NJ, USA); LPS from E. coli (055:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA); recombinant human CCL4 variant (S80T) was from Kingfisher Biotech, Inc (St Paul, MN, USA); anti-human CCR5-PE (CD195, Clone# J418F1), anti-human CCR1-APC (CD191, Clone# 5F10B29), anti-human CD4-PerCP (Clone# OKT4) and anti-human CD3-FITC (clone# SK7) were from Biolegend (San Diego, CA, USA); Indo-1-AM was from Molecular Probes (Eugene, OR).
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