p‐HLA tetramers were prepared by conjugating purified biotinylated p‐HLA monomers to streptavidin at a 8:1 monomer to streptavidin molar ratio. Streptavidin‐PE (Invitrogen, Waltham, USA) was added slowly onto the monomer at 1/10 of the volume required and incubated for 10 min at room temperature, 10 times. CD8+ T cell lines were tetramer stained for 1 h at room temperature. Cells were washed and surface stained with anti‐CD3‐BV480 (BD Biosciences), anti‐CD8‐PerCP‐Cy5.5 (BD Biosciences), anti‐CD4‐BUV395 (BD Biosciences) and live/dead fixable near‐IR dead cell stain (Life Technologies). Cells were fixed with 1% paraformaldehyde and acquired on the BD LSR Fortessa and were analysed using Flowjo v10 (BD Biosciences). The gating strategy is shown in Supplementary figure 1b .
Anti cd3 bv480
Manufactured by BD
Sourced in United States
Anti‐CD3‐BV480 is a fluorochrome-conjugated antibody that recognizes the CD3 cell surface antigen. CD3 is a key component of the T cell receptor complex and is expressed on the surface of T cells. The BV480 fluorochrome allows for detection and analysis of CD3-positive cells using flow cytometry.
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Lab products found in correlation
Anti cd8 percp cy5, by BD (4 mentions)
Live dead fixable near ir dead cell stain, by Thermo Fisher Scientific (4 mentions)
Anti cd4 buv395, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Anti cd4 bv650, by BD (2 mentions)
Cytofix cytoperm solution, by BD (2 mentions)
Anti tnf pe cy7, by BD (2 mentions)
Anti ifn γ bv421, by BD (2 mentions)
Streptavidin pe, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Anti cd107a fitc, by BD (1 mentions)
Anti mip1β apc, by BD (1 mentions)
Anti il 2 pe, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Fetal bovine serum (fbs), by Bovogen (1 mentions)
Anti cd19 apc h7, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
4 protocols using anti cd3 bv480
1
Staining CD8+ T Cells with pHLA Tetramers
2
Multiparametric Flow Cytometry of Activated CD8+ T Cells
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CD8+ T cell lines were stimulated with 1 μM of the cognate or the homologous peptide and were incubated for 4–5 h in the presence of GolgiPlug, GolgiStop and anti-CD107a-FITC (dilution 1:100) (all BD Biosciences). After stimulation, cells were surface stained for 30 min with anti-CD3-BV480 (1:100), anti-CD8-PerCP-Cy5.5 (1:50) and anti-CD4-BV650 (1:100) antibodies (all BD Biosciences) and Live/Dead Fixable Near-IR Dead Cell Stain (Life Technologies). Cells were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences) and then intracellularly stained with anti-IFN-γ-BV421 (1:100), anti-TNF-PE-Cy7 (1:100), anti-IL2-PE (1:100) and anti-MIP-1β-APC (1:100) antibodies (all BD Biosciences) for a further 30 min. Cells were acquired on the BD FACSymphony A3 system using the FACSDiva software (v.9.0.). Post-acquisition analysis was performed using FlowJo software (v.10). Cytokine detection levels identified in the no-peptide control condition were subtracted from the corresponding test conditions in all summary graphs to account for non-specific, spontaneous cytokine production.
3
Multiplex Intracellular Cytokine Staining Assay
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Intracellular cytokine staining (ICS) assay was completed as previously described.37 Briefly, CD8+ T cell lines were co‐cultured with peptide‐pulsed C1R‐HLA‐A*03:01 at a 1:2 stimulators: responders (APC: T cell) ratio and incubated for 5 h in the presence of GolgiPlug (BD Biosciences, Franklin Lakes, USA) and GolgiStop (BD Biosciences). Cells were stained with anti‐CD3‐BV480 (BD Biosciences), anti‐CD8‐PerCP‐Cy5.5 (BD Biosciences, Franklin Lakes, USA), anti‐CD4‐BUV395 (BD Biosciences) and live/dead fixable near‐IR dead cell stain (Life Technologies) for 30 min, fixed and permeabilised using BD Cytofix/Cytoperm solution (BD Biosciences) for 20 min and then intracellularly stained with anti‐IFN‐γ‐BV421 (BD Biosciences) as well as anti‐TNF‐PECy7 (BD Biosciences) for 30 min. Samples were acquired on a BD Fortessa and analysed using FlowJo v10 (BD Biosciences). The gating strategy is shown in Supplementary figure 1a .
4
Generating CD8+ T Cell Lines from PBMCs
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CD8+ T cell lines were generated as previously described25 (link),58 (link). In brief, PBMCs were incubated with 1 μM of individual peptide (NQK-Q8 or NQK-A8) and cultured for 10–14 days in RPMI-1640 supplemented with 2 mM MEM non-essential amino acid solution (Sigma-Aldrich), 100 mM HEPES (Sigma-Aldrich), 2 mM l -glutamine (Sigma-Aldrich), penicillin–streptomycin (Life Technologies), 50 mM 2-ME (Sigma-Aldrich) and 10% heat-inactivated fetal bovine serum (Bovogen). The cultures were supplemented with 10 IU IL-2 2–3 times weekly. CD8+ T cell lines were used fresh for subsequent analysis. For the double tetramer staining experiments 0.5 × 106 cells from the CD8+ T cell lines were stained with a single PE-conjugated tetramer (HLA-B*15:01-NQK-Q8 or HLA-B*15:01-NQK-A8) or double stained with both tetramers (PE-conjugated NQK-A8 and APC-conjugated NQK-Q8 tetramer) for 1 h at room temperature. Cells were washed and surface-stained with anti-CD3-BV480 (dilution 1:100), anti-CD8-PerCP-Cy5.5 (1:50), anti-CD4-BV650 (1:100), anti-CD14-APCH7 (1:200) and anti-CD19-APCH7 (1:100) antibodies (all BD Biosciences) and Live/Dead Fixable Near-IR Dead Cell Stain (Life Technologies). Cells were single-cell sorted into PCR plates (Eppendorf) using the BD Aria Fusion system.
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