Human IgD was purchased from Abcam (Cambridge, MA, USA). Anti-CD3 and CD28 antibodies were provided by T&L Biological Technology (Beijing, China). A770041 was purchased from Axon Medchem (Groningen, Netherlands). rhTNFR:Fc fusion protein was purchased from Guojian Pharmaceutical Company (Shanghai, China). Anti-mouse IgD antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-human CD69-PE-cy5, CD154-PE, CD4-FITC, CD25-APC, IL-4-APC, IFN-γ-PE-cy7, IL-17-PE, and FoxP3-PE, along with anti-mouse CD3e-PerCP-CyTM5.5, CD4-FITC, CD25-APC, CD154-PE, IFN-γ-PerCP-CyTM5.5, IL4-APC, IL-17-PE, and FoxP3-PE antibodies were provided by BD Pharmingen (San Diego, CA, USA). Anti-Lck, anti-phospho-ZAP70, anti-phospho-Lck, anti-ZAP70, and anti-β-actin were purchased from Affinity Biosciences, Sigma and Abcam.
Il 4 apc
Manufactured by BD
Sourced in United States
IL-4-APC is a fluorescently-labeled antibody that binds to the human interleukin-4 (IL-4) protein. It is commonly used in flow cytometry applications to detect and quantify IL-4 expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Il 17 pe, by BD (3 mentions)
Foxp3 pe, by BD (3 mentions)
Ifn γ fitc, by BD (3 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Brefeldin a, by BD (2 mentions)
Efluor 780, by Thermo Fisher Scientific (2 mentions)
Gata 3 pe, by Thermo Fisher Scientific (2 mentions)
Ifng fitc, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (2 mentions)
Il 4 apc, by Thermo Fisher Scientific (2 mentions)
Ebio13a, by Thermo Fisher Scientific (2 mentions)
Ebio17b7, by BD (2 mentions)
Il 17f pe, by BD (2 mentions)
Il 5 pe, by BioLegend (2 mentions)
Tnfa alexafluor700, by BD (2 mentions)
Il 13 fitc, by Thermo Fisher Scientific (2 mentions)
Pvm13 1, by BD (2 mentions)
Il 17a efluor450, by Thermo Fisher Scientific (2 mentions)
Il 13 pe, by Thermo Fisher Scientific (2 mentions)
Il 10 apc, by BD (2 mentions)
12 protocols using il 4 apc
1
Immunological Analysis of T Cell Activation
2
Immunophenotyping of Th17 and Treg Cells
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For FCM analysis, 1 × 106 cells per sample were used, and the cells were labeled with CD4-BB515. After permeabilization and fixation, Th17 and Treg cells were incubated with IL-17A-BV421 or Foxp3-PE, respectively. For Th17 cells, the cells were stimulated for 4–6 h in an incubator (37°C, 5% CO2) with a leukocyte activation cocktail prior to antibody incubation. The cells were detected by FLow cytometry (BD LSRFortess, Franklin Lakes, NJ, United States) and analyzed utilizing the FlowJo software (Tree Star, Inc. San Carlos, CA, United States). To detect CD3, CD8, Th1 and Th2 cells, CD3-APC-Cy7, CD8-APC, interferon (IFN)-γ-PE, and IL-4-APC from BD Bioscience were used.
Cytokines in serum or culture supernatants were measured using a commercially available ELISA kit (eBioscience, San Diego, CA, United States) according to the manufacturer’s protocols.
Cytokines in serum or culture supernatants were measured using a commercially available ELISA kit (eBioscience, San Diego, CA, United States) according to the manufacturer’s protocols.
3
Intracellular Cytokine Staining Protocol
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Following stimulation assays and surface staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm kit (BD Biosciences) as per manufacturer’s instructions. Cells were then stained for intracellular cytokines from the following list: IL-4-APC (BD Pharmingen), TNFα-APC-Cy7 (MAb11; BioLegend), IFNγ-BV711 (B27; BD Horizon) IL-17A-BV786 (N49-653; BD Horizon).
4
Profiling Lung T Cell Activation and Cytokines
5
Multiparametric Flow Cytometry for Cytokine and Transcription Factor Analysis
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For cytokine staining, cells were restimulated with 20 nM PMA and 1μM ionomycin for 4 hours, and 5μg/ml brefeldin A was added during the last 2 hours of restimulation. Cells were stained with the viability dye eFluor780 (eBiosciences; Cat # 65-0865-14), and then fixed in 4% paraformaldehyde for 8 minutes at room temperature. Cytokine staining was done in permeabilization buffer containing 0.05% saponin. For transcription factor staining, unstimulated cells were fixed, permeabilized, and stained using the FoxP3 Staining Kit (eBiosciences; Cat # 00-5523-00). Samples were analyzed with a flow cytometer (BD LSR II). Mouse antibodies: IL-13-PE (eBiosciences; eBio13A), IL-4-APC (eBiosciences; 11B11), IL-5-PE (Biolegend; TRFK5), IFNg-FITC (eBiosciences; XMG1.2), TNFa-AlexaFluor700 (BD Biosciences; MP6-XT22), and GATA-3-PE (eBiosciences; E50-2440). Human antibodies: IL-13-FITC (eBiosciences; PVM13-1), IL-4-APC (BD Biosciences; 8D4-8), IL-17A-eFluor450 (eBiosciences; eBio17B7), and IL-17F-PE (BD Pharmingen; 079-289).
6
Detecting Intracellular Immune Markers in HIV-Infected Cells
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To measure intracellular activation markers, cells were permeabilized for 30min with PermWash at RT (BD Pharmingen) and subsequently stained with p24-PE (Beckman Coulter) and IFN-γ-FitC, IL2-PerCp-Cy5.5, IL-4-APC, TNF-α-PE-CF594, Mip-1β-AlexaFluor700 (all from BD Bioscience) for 30min at 4°C at a pre-determined dilution. Next, these cells were washed once with PermWash and taken up in FACS buffer (PBS+ 2%FCS) after which they were analyzed using a FACS Canto II machine. For determining CCR5 surface expression, T-cells obtained from our T-cell outgrowth model were fixed in 3.7% formaldehyde before HIV-1 infection and stored for no more than 1 week at 4°C in FACS buffer. These cells were stained in FACS buffer with CCR5 PE-Cy7 (Biolegend) for 30min at RT, washed with FACS buffer and measured. Shown in histograms are cytokine producing T-cells with samples taken 5 and 7 days after HIV-1 infection with the optimal time point being determined by the percentage of live cells (>50%) and level of HIV-1 infection, which varied per donor and per virus. Gating strategies and flow cytometric controls are represented (S3A Fig and S3B Fig , respectively).
7
Multiparameter Flow Cytometry of PBMCs
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PBMCs were isolated from whole blood cells using Ficoll media purchased from Haoyang Bio-Manufacture CO., LTD (Tianjin, China). Staining fixable viability stain 780 (BD#565388) was added into the cell suspension at 1:1000, followed by an incubation with human Fcblock (BD#564220). Surface markers were stained using CD3-PerCP-Cy5.5 (BD#560835), CD4-PE-Cy7 (BD#560649), CD8-BV510 (BD#563256), CD25-PE (BD#557138), CD127-AF647 (BD#558598), CD45RA-APC (BD#550855) and CCR7-FITC (BD#560548). For intracellular staining, cells were fixed and permeablized using BD CytoFix/CytoPerm Kit (BD#554715) and then the cytokines were stained using IL-4-APC (BD#560671), IFN-γ-FITC (BD#554700), TNF-α-BV421 (BD#562783), and IL-17-PE (BD#560487), before the final flow cytometry assay using BD CantoII. Raw data of flow cytometry was analyzed by FlowJo software.
8
Multiparametric Flow Cytometry Analysis of Murine Immune Cells
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Mouse spleen and lymph nodes (mesenteric for the T-cell populations and inguinal for B-cell populations) were harvested and crushed to obtain a single-cell suspension. In the spleen, red blood cells were removed by a red blood lysis buffer (Invitrogen, Life Technologies, USA). A total of 1 × 106 cells were transferred to 96-well plates and stimulated for 5 h with RPMI, 5% FCS, 1% penicillin/streptavidin, phorbol-12-myristate-13-acrylate (50 ng/ml), and ionomycin (1 µg/ml) at 37°C, 5% CO2. Brefeldin A and monensin (1 mg/ml, BD Biosciences, USA) were also added 2 h after stimulation. Cells were labeled with surface markers (CD3-FITC, CD4-PerCP-Cy5.5, CD8a-PE-Cy7, CD25-Brilliant Vio510; CD19-PerCP vio770, CD9 PE-vio770, BD Biosciences and Miltenyi Biotec, France) with the Fc blocking antibody anti-CD16/CD32 (BD Biosciences). Cells were fixed and permeabilized with a Fixation/Permeabilization Solution Kit (BD Biosciences) to perform intracellular labeling (Fox-P3-PE, IL-4-APC, IFN-γ-APC-Cy7, BD Biosciences). Cells were analyzed by flow cytometry on a BD FACSCanto™ II Cell Analyzer (BD Biosciences). Data were acquired with Diva 8.0 software and analyzed with FlowJo Software v10 (TreeStar, Williamson Way, USA).
9
Multiparametric Flow Cytometry for Cytokine and Transcription Factor Analysis
10
Characterization of T-cell Cytokine Profile
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T cells were stimulated with Dynabeads Human/Mouse T‐Activator CD3/CD28 (Gibco, Invitrogen, Carlsbad, CA). After 72 h of culture, GolgiPlug (BD Pharmingen, San Diego, CA, USA) was added for 4 h. Cells were harvested for surface staining as described above. Intracellular staining was carried out by using a fixation/permeabilization kit (BD Bioscience) after resuspension according to the manufacturer's instructions. For human samples, IL‐2–V450, IFN‐γ–BV510, IL‐17–PE, IL‐4–APC, and IL‐10–PE (BD Pharmingen) were added and incubated for 20 min at room temperature. For mouse samples, IFN‐γ–PE (BD Pharmingen) was added and incubated for 30 min at 4 °C. Detailed antibody information was listed in Table S3 (Supporting Information).
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