NKG2A− and NKG2A+ subpopulations were stimulated in U-bottomed 96-well plate (NUNC) with 400 ng/mL of plate-bound anti-CD3 mAb (produced by OKT3, ATCC CRL-8001, RRID:CVCL_2665) for 5 hours in the presence of 10 µg/mL of Brefeldin A (Sigma-Aldrich). After fixation with PBS 4% paraformaldehyde (VWR) for 10 minutes, cells were washed in PBS 0.1% BSA 0.1% Saponin (Sigma-Aldrich), centrifuged then stained with anti-human TNF-α-FITC (BioLegend, Mab11, RRID:AB_315258) and IFN-γ-APC (BioLegend, B27, RRID:AB_315443) or IL-2-APC (BioLegend, MQ1-17H12, RRID:AB_315098) mAbs for 30 minutes in PBS 0.1% BSA 0.1% Saponin. For the CD107a degranulation assay, cells were stimulated for 3 hours without Brefeldin A in the presence of anti-human CD107a-AF647 mAb (BioLegend, H4A3, RRID:AB_1227506) added at the beginning of stimulation. Stained cells were acquired in the singlet-cell gate on a BD FACSCanto II flow cytometer using the same software as above.
Il 2 apc
Manufactured by BioLegend
IL-2 APC is a fluorescently labeled antibody that binds to the interleukin-2 (IL-2) protein. IL-2 is a cytokine that plays a crucial role in the activation and proliferation of T cells, which are important for the adaptive immune response. The APC fluorophore attached to the antibody allows for the detection and quantification of IL-2-expressing cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ apc, by BioLegend (3 mentions)
Brefeldin a, by BioLegend (3 mentions)
Ifn γ pe, by BioLegend (3 mentions)
Il 17 bv421, by BioLegend (2 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Saponin, by Merck Group (1 mentions)
U bottomed 96 well plate, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Avantor (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Anti human tnf α fitc, by BioLegend (1 mentions)
Saponin, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd4 fitc, by BioLegend (1 mentions)
Igg1 pe, by BioLegend (1 mentions)
B220 fitc, by BioLegend (1 mentions)
Cd5 pe cy7, by BioLegend (1 mentions)
Il 17 pe, by Miltenyi Biotec (1 mentions)
Anti fcγrii fcγriii 2.4g2, by BD (1 mentions)
Igm fitc, by BioLegend (1 mentions)
Plus brefeldin a, by Thermo Fisher Scientific (1 mentions)
8 protocols using il 2 apc
1
Functional Characterization of NKG2A+ Cells
2
B Cell Immunophenotyping and Cytokine Analysis
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B cells were washed once in cold PBS containing 0.1% BSA (FACS buffer) before blocking with anti-FcγRII/FcγRIII (2.4G2, BD Pharmingen, San Diego, CA). Stainings were performed on ice using conjugated mAbs (eBioscience, San Diego, CA, if not stated otherwise), diluted 1:300 in FACS buffer for surface marker and 1:200 for intracellular cytokine staining followed by incubation for 20 min. After washing with FACS buffer, cells were analysed on a FACS Canto II (BD) using FlowJo software (Tree star, Ashland, OR). The following Abs were used: B220-FITC (#11-0452-86), CD5-PE-Cy7 (#25-0051-81), CD1d-PE (#12-0011-81), CD138-APC (#142506, Biolegend), IgM-FITC (#11-5890-85), IgG1-PE (#12-4015-82), CD4-FITC (#11-0041-82), IL-2-APC (#17-7021-81), IFNγ-APC (#17-7311-82) and IL-10-PerCP (#45-7101-80). Abs against TNF−α−PE (#130-092-245) and IL-17-PE (#130-094-296) were from Miltenyi Biotec. For intracellular staining, the fixation and permeabilization kit (Plus Brefeldin A; eBioscience, Cat. no. 88-8823-88) was used according to manufacturer's recommendation.
3
Comprehensive T Cell Activation Profiling
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Peptide stimulated cells were washed and extracellularly stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Afterwards, samples were fixed and permeabilized for 30 min at 4°C using FoxP3 transcription factor staining buffer set (eBioscience). Following, intracellular staining was performed for CD3 BV650, CD4 PerCp-Cy5.5, CD8 BV510, CD137 PE, CD154 BV421, IL-2 APC, IFNγ BV605, and TNFα AF700 (Biolegend). Stained cells were then transferred into a 96-well plate and measured at a CytoflexLX (Beckman Coulter). Flow cytometry data was analyzed using FlowJo software version 10.6.2 (BD). Reactive T cells were defined as CD154+CD137+CD4+ or CD137+CD8+ T cells >0.005% within total CD4+ or CD8+ T cells and with a threshold of ≥1.2-fold signal above the background control. This threshold corresponds to the range in which 95% of all negative samples are. Unspecific activation of cells was excluded by subtracting the background signal of the DMSO stimulated negative control sample from the peptide stimulated samples. Single, double (dp), or triple (tp) cytokine producing reactive T cell subsets were analyzed using Boolean combination gates (see Supplementary Figure 1
4
Pneumococcal Infection: T-cell Immune Profiling
5
Cytokine Production Profiling of Activated T Cells
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For functional assays on cytokine production by T cells, thawed isolated PBMCs were stimulated for 16 h at 37 °C in a 5% CO2 atmosphere with anti-CD3/CD28 (1 μg/mL) in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β-mercaptoethanol). For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson) and previously titrated concentration of CD107a-PE. After stimulation, cells were stained with LIVE-DEAD Aqua (ThermoFisher Scientific) and surface mAbs recognizing CD3 PE- Cy5, CD4 AF700, and CD8 APC-Cy7 (Biolegend, San Diego, CA, USA). Cells were washed with stain buffer, and fixed and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Cells were next stained with previously titrated mAbs recognizing IL-17 BV421, TNF BV605, IFN-γ FITC, IL-2 APC, or granzyme-B BV421 (all mAbs from Biolegend). Then, a minimum of 100,000 cells per sample were acquired on a Attune NxT acoustic cytometer (ThermoFisher).
6
Multiparametric Flow Cytometry Analysis
7
Characterizing Anti-CD19 CAR T Cell Responses
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Flow cytometry was performed using a FACSAria II cell counter (BD Biosciences). T cells were assessed for the surface presentation of epitopes with fluorescently labeled monoclonal antibodies for CD69 (BioLegend). In parallel, anti‐CD19 CAR expression was measured using Flow cytometry. First, anti‐CD19 CAR expression was measured with commercial bio‐sCD19‐Fc (ACROBiosystems), followed by staining with secondary APC‐conjugated SA antibody (BioLegend). Anti‐CD19 CAR expression was measured with sCD19‐SA, followed by a secondary PE‐conjugated anti‐SA antibody (BioLegend), and anti‐CD19 CAR expression was directly measured with FITC‐sCD19‐SA. To assess cytokines produced by CAR‐T cells, cells were incubated in 96‐well U‐bottom plates at 105 cells/100 μl media/well for 24 h in the presence of 60 μg/ml sCD19‐SA with 5 μg/ml Brefeldin A (BioLegend), and the hGM‐CSF‐SA fusion protein (prepared in our laboratory) was added to the medium alone as a negative control. Before permeabilization with ice‐cold methanol, the cells were fixed in 1.5% formaldehyde and then incubated with antibodies against FITC‐TNF‐α, PE‐IFN‐γ, and APC‐IL‐2 (all from BioLegend) for flow cytometric analysis. A total of 104 cells were counted by flow cytometry for each experiment.
8
Multiparametric Flow Cytometry Analysis
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Anti-human mAbs included PE-Cy7-CD3 (Biolegend), PerCP-Cy5.5-CD8 (Biolegend), APC-IL-17 (eBioscience), PE-IFN-γ (Biolegend), APC-IL-2 (Biolegend), PE-Cy7-CD19 (Biolegend), FITC-Zombie (Biolegend), APC-fire750-CD69 (Biolegend), PerCP-Cy5.5-TGF-β (Biolegend), APC-IL-10 (Biolegend), PE-GrB (eBioscience). After stimulation, PBMC were harvested and first stained with surface antibodies followed by fixation/permeabilization (Cytofix/Cytoperm kit, BD Biosciences) and subsequent intracellular staining, as previously described [13 (link)].
Flow cytometry was performed in NovoCyte D2060R (ACEA Biosciences Inc). NovoEXpress software (San Diego, CA, USA) was used for analysis. Flow cytometry characterization of lymphocyte subsets is presented in Additional file1 : Figure S1.
Flow cytometry was performed in NovoCyte D2060R (ACEA Biosciences Inc). NovoEXpress software (San Diego, CA, USA) was used for analysis. Flow cytometry characterization of lymphocyte subsets is presented in Additional file
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