For the identification of CD4+ Th cell subsets and CD8+ Tc cells, we used cytoplasmic cytokine staining method. Briefly, whole blood were diluted to 1:1 with saline solution and incubated with phorbol-12-myristate 13-acetate (PMA) (25 ng/ml), ionomycin (1 μg/ml) and Golgi Stop brefeldin A (10 μg/ml) (all from Sigma Aldrich, St. Louis, Missouri, USA) for 5 h at 37°C in 5% CO2 milieu. The following monoclonal antibodies were used for cell surface staining: CD4-PE-Cy5 or CD8-PE-Cy5 (both from Beckman Coulter). The cells were then fixed and permeabilized with Intraprep™ permeabilization reagent (Beckman Coulter) according to the manufacturer’s instructions, and intracellular cytokines were stained with the combination of interferon (IFN)-γ-FITC, interleukin (IL)-4-PE, IL-10-PE (all from BD Biosciences) and IL-17-PE (R&D Systems, Minneapolis, MN, USA) monoclonal antibodies. Measurements were performed and data were analyzed on Coulter FC500 flow cytometer (Beckman Coulter) equipped with Kaluza 1.2a software. IgG1-FITC (BD Biosciences) and IgG1-PE (R&D Systems) isotype-matched antibodies were used during the identification. Cells were quantified as their percentage in the CD4+ or CD8+ lymphocyte population.
Il 10 pe
Manufactured by BD
Sourced in United States
IL-10-PE is a fluorescently labeled antibody that binds to the human interleukin-10 (IL-10) protein. It is designed for use in flow cytometry applications to detect and quantify IL-10 expression in cells.
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Lab products found in correlation
Ifn γ fitc, by BD (5 mentions)
Facscalibur, by BD (4 mentions)
Ionomycin, by Merck Group (3 mentions)
Perm wash, by BD (3 mentions)
Cd25 apc, by BD (3 mentions)
Cytofix cytoperm, by BD (3 mentions)
Il 17 pe, by BD (2 mentions)
Il 4 pe, by BD (2 mentions)
Cd4 percp, by BD (2 mentions)
Lsr fortessa instrument, by BD (2 mentions)
Fix perm buffer, by BD (2 mentions)
Cd3 ecd, by Beckman Coulter (2 mentions)
Ifnγ v450, by BD (2 mentions)
Cd4 qdot655, by Thermo Fisher Scientific (2 mentions)
Vα24 fitc, by Beckman Coulter (2 mentions)
Withperm wash buffer, by Thermo Fisher Scientific (2 mentions)
Pbs57 cd1d tet apc, by Beckman Coulter (2 mentions)
Cd8 qdot 605, by Thermo Fisher Scientific (2 mentions)
Tnf alexa700, by BD (2 mentions)
Il 4 pe cy7, by BD (2 mentions)
28 protocols using il 10 pe
1
Flow Cytometric Identification of Lymphocyte Subsets
2
Cytokine Profiling of Activated Splenocytes
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Splenocytes were obtained from mice and incubated at 37 °C for 6 h in 48-well plates coated with anti-CD3 and anti-CD28 antibodies (1 mg ml−1 each; eBioscience, San Diego, CA, USA). Cells were stained for 30 min at 4 °C with surface-specific antibodies (anti-CD3-allophycocyanin, anti-CD4-FITC and anti-CD8-PE–Cy5; BD Biosciences, Franklin Lakes, NJ, USA) and then fixed and permeablized through incubation for 10 min in 4% paraformaldehyde at room temperature. Cells were incubated for 30 min at room temperature with antibodies specific for candidate cytokines (anti-IFN-γ–PE, -IL-17–PE, -IL-4–PE and -IL-10-PE; BD Biosciences) and then analyzed on a FACSCalibur flow cytometer (BD Biosciences) using the CellQuest software (CellQuest, Largo, FL, USA).
3
In Vitro Differentiation and Analysis of TH17 Cells
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In vitro differentiated and purified blood memory TH17 cells were restimulated during 3 h with PMA and ionomycin using the Cell Stimulation Cocktail (eBioscience) and then stained with Alexa647-IL26, Alexa488-IL17A, Alexa568-IL22 and Alexa750-IFNγ probes using PrimeFlow RNA Assay (eBioscience) according to the manufacturer’s protocol. In some experiments, intracellular cytokine staining of re-stimulated TH17 cells and blocked for secretion by Brefledin A after 1 h was performed using anti-human IL-9 PeCy7 (BioLegend, 1/20), IL-10 PE (BD Biosciences, 1/20), IL-13 BV421 (BioLegend, 1/20) and IL-21 PE (BioLegend, 1/20) antibodies before proceeding with the hybridization step of the Prime-Flow Assay. Cells were acquired on a FACS LSR II SORP (BD Biosciences) and analyzed with FlowJo_v10.7.1. A detailed gating strategy for this analysis is shown in Supplementary Fig. 8 .
4
Multiparameter Analysis of Tonsillar Lymphocytes
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Tonsillar MNCs were treated with Brefeldin A (eBiosciences) for 4 h before harvesting cells in order to block cytokine secretion. MNCs were stained with fluorescence labeled anti-human CD4, followed by fixation and permeabilization and staining intracellularly with fluorescence labeled anti-human Foxp3, IL-17A, IFNγ, IL-10, RORγt, and Ki67 in different combinations. Stained cells were acquired on BD Celesta or BD Canto II and data was analyzed by Flowjo. Anti-human CD4-PECy7, IL-17A-Alexa Fluor 647, Foxp3-Alexa Fluor 488, IL-10-PE, and RORγt-PE FACS antibodies were from BD Biosciences. Other antibodies were purchased from BioLegend.
5
Comprehensive Immune Cell Profiling
6
Splenic Leukocyte Profiling by Flow Cytometry
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48 hours following the final behavioral test, spleens were harvested and a single cells suspension of splenocytes prepared for flow cytometry analysis as we have described previously23 ,26 (link). Splenocytes (106) were stained for markers of dendritic cell (DC) maturation and function—CD11c- PerCP-Cy5, MHCII-FITC, CD80-PE, and CD86-APC—or regulatory T cells—CD3-APC, CD4-FITC, CD25-PE-Cy7, and intracellular IL-10-PE (BD Pharmingen, San Diego, CA, USA; eBioscience, San Diego, CA, USA). Following surface staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm before staining for intracellular markers. Data were acquired with FACSCanto (Becton Dickinson, Oakville, ON, Canada) and analysed using FlowJo (TreeStar, Ashland, OR, USA) with doublets and cell debris excluded by FSC and SSC gating. Gating strategies are shown in Fig. S1 .
7
IL-10 Production in B-cells After CD40L Activation
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PBMCs samples were thawed from liquid nitrogen on the same day as the staining and 3 × 106 PBMCs were stained with Live/Dead (Life Technologies Ltd, Paisley, UK), anti-CD20-AlexaFluor780 and CD86-PE (all eBioscience, Hatfield, UK) for 30 min/4 °C. 1 × 106 PBMCs from patient samples were rested overnight and the next day cells were cultured with 0.5 × 105 plate bound human-CD40L-transfected and non-transfected mouse L fibroblast cells (X-ray irradiated for 30 min 9,045 cGy) for 72 h. Intracellular IL-10-PE (BD) was measured in CD20+B-cells after CD40L activation.
8
Multiparametric Flow Cytometry of Immune Cells
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Cells were incubated with Fc Block (1 µg/106 cells; 2.4G2; BD Biosciences) for 15 min. Extracellular staining was performed for 30 min on ice using: CD4‐BV521 (RM4‐5; BioLegend, San Diego, CA, USA), CD45‐BV510 (30‐F11; BioLegend), CD3‐APC (17.A2, BioLegend), CD25‐PE‐Cy7 (PC61; BioLegend), CD8‐PerCPCy5.5 (53‐6.7; BioLegend), B220 (RA3‐6B2; BD Biosciences), CD11b‐PE‐Cy7 (M1/70; BioLegend), Ly6C‐PE (HK1.4; BioLegend), Gr1‐APC‐Cy7 (RB6‐8C5, BioLegend), IA/IE‐BV421 (M5/114.15.2, BioLegend), F4/80‐FITC (BM8, BioLegend), CD11c‐PerCPCy5.5 (N418, BioLegend).
After staining for extracellular proteins, cells were fixed in 4% PFA and permeabilised using 0.1% saponin buffer containing 0.1% bovine serum albumin. Intracellular cytokines were detected with IFNγ‐BV421 (XMG 1.2, BioLegend), IL‐10‐PE (54902, BD Bioscience), and IL‐17A‐AF647 (TC11‐18H10, BioLegend).
Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analysed using FlowJo software version 10.6 (Treestar Inc., Ashland, OR, USA).
After staining for extracellular proteins, cells were fixed in 4% PFA and permeabilised using 0.1% saponin buffer containing 0.1% bovine serum albumin. Intracellular cytokines were detected with IFNγ‐BV421 (XMG 1.2, BioLegend), IL‐10‐PE (54902, BD Bioscience), and IL‐17A‐AF647 (TC11‐18H10, BioLegend).
Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analysed using FlowJo software version 10.6 (Treestar Inc., Ashland, OR, USA).
9
Flow Cytometry Immunophenotyping Protocol
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For flow cytometry analyses cells were stained with antibodies to detect surface expressed CD4 (CD4 Alexa 488; CD4 APC; CD4 Percp [BD Biosciences]) and intracellular expressed RORγT, IL‐10, IL‐17A, and IFN‐γ (RORγt PE, IFN‐γ FITC, IL‐17A Percp, IL‐10 PE‐ BD biosciences). Staining was performed with Fix/Perm and Perm/Wash from BD Biosciences according to the manufacturer's protocols. To monitor cytokine expression by flow cytometry, cells were incubated with the corresponding stimuli and stimulated for 4 h in the presence of PMA (50 ng/mL), Ionomycin (500 ng/mL) and 1.5 μL/1 mio cells Golgi‐StopTM (BD Biosciences) before staining. Cells were analyzed using FACS Calibur from BD Bioscience and data analysis was performed with FlowJo software from Treestar (San Carlos).
10
Multiparameter Flow Cytometry Analysis
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