SIV-specific CD8+ T cells were quantified in mononuclear cells isolated from blood and LN by flow cytometric intracellular cytokine analysis, as previously described in detail45 (link). Briefly, mixes of sequential (11 amino acid overlapping) 15-mer peptides (AnaSpec) spanning the SIVmac239 Gag, Env, Pol, Nef, Rev/Tat, Vif/Vpr/Vpx proteins were used as antigens in conjunction with anti-CD28 (BD Biosciences) and anti-CD49d co-stimulatory mAb (BD Biosciences). Cells were incubated at 37 °C with peptide mixes/pools and co-stimulatory antibodies for 1 hr, followed by an additional 8 hr. incubation in the presence of Brefeldin A (5 μg/ml, Sigma-Aldrich). Co-stimulation in the absence of peptides served as background control. Cells were stained with fluorochrome-conjugated monoclonal antibodies [CD3 (SP34-2), CD4 (L200), CD8a (SK1), IFN-γ (B27)(BD Biosciences), TNF (MAB11, eBioscience), CD69 (FN50, BD Biosciences)], and data were collected on an LSRII (BD Biosciences) and analyzed using the FlowJo software program (version 8.8.7; Tree Star, Inc.). Response frequencies to each peptide mix were defined by the intracellular expression of TNF and/or IFN-γ by CD3+, CD4−, CD8+ T cells after subtraction of background. Response frequencies to each peptide mix were added to obtain total SIV-specific response frequencies.
Cd69 fn50
Manufactured by BD
Sourced in Germany
CD69 (FN50) is a lab equipment product. It is a cell surface molecule that is expressed on activated T cells, B cells, and natural killer cells. The core function of CD69 is to serve as an early activation marker for these immune cell types.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ b27, by BD (2 mentions)
15 mer peptides, by AnaSpec (2 mentions)
Anti cd49d co stimulatory mab, by BD (2 mentions)
Tnf mab11, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Anti cd28, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
7 aminoactinomycin d viability dye, by Merck Group (1 mentions)
Vβ11 c21, by Beckman Coulter (1 mentions)
Tcr vβ repertoire kit, by Beckman Coulter (1 mentions)
Cd4 sk3, by BD (1 mentions)
247 ilb, by R&D Systems (1 mentions)
Cd8 sk1, by BD (1 mentions)
Cd45ro uchl1, by BD (1 mentions)
Cx3cr1 2a9 1, by BioLegend (1 mentions)
Cd57 hnk 1, by BioLegend (1 mentions)
Cd27 m t271, by BD (1 mentions)
Ccr7 3d12, by BD (1 mentions)
Aqua live dead viability dye, by Thermo Fisher Scientific (1 mentions)
Anti granzyme b gb11, by BD (1 mentions)
7 protocols using cd69 fn50
1
Quantification of SIV-specific CD8+ T Cells
2
Quantification of SIV-specific CD8+ T Cells
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SIV-specific CD8+ T cells were quantified in mononuclear cells isolated from blood and LN by flow cytometric intracellular cytokine analysis, as previously described in detail45 (link). Briefly, mixes of sequential (11 amino acid overlapping) 15-mer peptides (AnaSpec) spanning the SIVmac239 Gag, Env, Pol, Nef, Rev/Tat, Vif/Vpr/Vpx proteins were used as antigens in conjunction with anti-CD28 (BD Biosciences) and anti-CD49d co-stimulatory mAb (BD Biosciences). Cells were incubated at 37 °C with peptide mixes/pools and co-stimulatory antibodies for 1 hr, followed by an additional 8 hr. incubation in the presence of Brefeldin A (5 μg/ml, Sigma-Aldrich). Co-stimulation in the absence of peptides served as background control. Cells were stained with fluorochrome-conjugated monoclonal antibodies [CD3 (SP34-2), CD4 (L200), CD8a (SK1), IFN-γ (B27)(BD Biosciences), TNF (MAB11, eBioscience), CD69 (FN50, BD Biosciences)], and data were collected on an LSRII (BD Biosciences) and analyzed using the FlowJo software program (version 8.8.7; Tree Star, Inc.). Response frequencies to each peptide mix were defined by the intracellular expression of TNF and/or IFN-γ by CD3+, CD4−, CD8+ T cells after subtraction of background. Response frequencies to each peptide mix were added to obtain total SIV-specific response frequencies.
3
Characterization of CD1d-Reactive T Cells
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Enriched CD3+ CD1d–α-GalCer tetramer+ cells were stained with Vδ1 (clone A13 supernatant, which can bind to Vδ1 when incorporated in hybrid Vδ1-Jα-Cα TCR chains, was produced in L. Moretta’s laboratory), anti–mouse IgG (clone Poly 4053; BioLegend), 5% normal mouse serum, and then with antibodies specific for αβTCR (clone T10B9.1A-31; BD), CD3ε (clone UCHT1; BD), CD8α (SK1; BD), CD4 (RPA-T4; BD), Vβ11 (C21; Beckman Coulter), CD69 (FN50; BD), γδTCR (11F2; BD), and CD161 (191B8; Miltenyi Biotec). Cells were costained with 7-aminoactinomycin D viability dye (Sigma-Aldrich) and with human CD1d tetramers (produced in-house), as previously described (Uldrich et al., 2013 (link)). TCR-Vβ repertoire analysis was performed using a TCR-Vβ repertoire kit (Beckman Coulter). Cells were analyzed using an LSR Fortessa (BD), and data were analyzed using FlowJo software (Tree Star Inc.).
4
Multiparametric Flow Cytometry for T Cell Phenotyping
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T cell phenotype was assessed by flow cytometry (LSRFortessa, BD) using fluorochrome-conjugated antibodies specific for CD3 (clone UCHT1; BD), CD4 (SK3; BD), CD8 (SK1; BD), CD45RO (UCHL1; BD), CX3CR1 (2A9-1; BioLegend), CD57 (HNK-1; BioLegend), CD27 (M-T271; BD), CD28 (CD28.2; BioLegend), CD69 (FN50; BD), and CCR7 (3D12; BD). Viable cells were gated using Live/Dead Aqua viability dye (Invitrogen). For induction and detection of intracellular cytokines, cells were cultured overnight with 20ng/ml recombinant human IL-15 (247-ILB; R&D Systems) or medium control (RPMI 1640 [Gibco], supplemented with 10% fetal bovine serum [Gemini Bio-Products], 1% L-glutamine [Gibco], and 1% penicillin/streptomycin [Gibco]), then treated with brefeldin A (GolgiPlug, BD) for 6h prior to harvest. After Live/Dead and surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD) for 20 min on ice and stained for 40 minutes on ice with anti-IFNγ (B27, BD) and anti-TNF (MAb11, BD). For intracellular accumulation of cytolytic molecules, cells were treated with IL-15-supplemented or control medium for 48 hours, then harvested, stained with Live/Dead and surface antibodies, treated with Cytofix/Cytoperm, stained with anti-granzyme B (GB11; BD) and anti-perforin (B-D48; BioLegend).
5
Activation of Human Granulocytes Analysis
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To analyze the activation of human granulocytes, eosinophils were identified as Siglec8‐positive (7C9; Bio Legend) and CD16‐negative (3G8; Bio Legend) and Annexin V‐negative (120F; IQP). Neutrophils were identified as CD16‐positive (3G8; Bio Legend) and Annexin V‐negative. A total of 50 000 granulocytes were incubated with mAbs for 30 minutes at 4°C, and 10 minutes with Annexin V at 4°C. An assessment of the activation of cell‐surface markers was made by the use of mAbs against the following molecules: CD63 (H5C6; Bio Legend), CD69 (FN50; BD Pharmingen). Cells were washed in PBS containing 0.5% BSA. Data acquisition was done using FACSCanto II (BD Biosciences).
6
Phenotypic Analysis of Activated T Cells
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After 24 h, 48 h, 72 h, and 6 d cultured cells were harvested, magnetically separated from stimulation beads and washed with FACS buffer (phosphate buffered saline with 2% fetal calf serum). Viability was determined by flow cytometry after 72 h and 6 d using Nicoletti buffer according to the protocol described elsewhere [47 (link)]. Surface markers were stained with anti-human antibodies against CD28 (CD28.2, BD, Heidelberg, Germany), CD137 (4B4, eBioscience, Frankfurt a.M., Germany), CD25 (M-A251, BD), CD69 (FN50, BD), CD16 (3G8, BD Bioscience), CD32 (FUN-2, BioLegend, San Diego, CA, USA), CD64 (10.1, BD Bioscience), CD152 (BN13, BD Bioscience), and CD279 (EH12.2H7, BioLegend). Isotypes and quiescent cells were stained as negative controls. The data were recorded on a FACS Calibur cytometer or an LSRFortessa flow cytometer (both BD), and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
7
Multiparameter Immunofluorescence Imaging
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Cryosections were fixed with 4 % paraformaldehyde and blocked with avidin/biotin blocking reagent (Vector Laboratories) and protein-blocking reagent (Roth). The sections were incubated with antibodies against CD4 (RPA-T4), CD8 (RPA-T8, both BD; polylonal, abcam), E-Cadherin (36/E, BD), Foxp3 (236A/E7, eBioscience), αE integrin (ab129202, Abcam), CD11c (BU15, AbD Serotec), CD69 (FN50, BD), CD123 (6H6, eBioscience), CD141 (Qbend/40, AbD Serotec), vedolizumab and etrolizumab surrogate followed by fluorescent-or biotin-labeled secondary antibodies (Vectorlabs and Merck). If applicable, slides were treated with Dylight488-or Cy3-conjugated streptavidin (Biolegend). Nuclei were counterstained with Hoechst reagent (molecular probes) and samples were analyzed by fluorescence confocal microscopy (LSM SP8, Leica). Single and double positive cells in at least three high power fields were counted.
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