Il 10 jes5 16e3

Manufactured by Thermo Fisher Scientific

The IL-10 (JES5-16E3) is a laboratory reagent used for the detection and quantification of the cytokine interleukin-10 (IL-10) in biological samples. It is a monoclonal antibody that specifically binds to IL-10. The IL-10 (JES5-16E3) can be used in various immunoassay techniques, such as ELISA, to measure IL-10 levels in cells, tissues, or body fluids.

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10 protocols using il 10 jes5 16e3

Multiparametric Flow Cytometry Analysis

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Single cell suspensions of spleen or draining lymph node were immunostained using various combinations of the following fluorescence-conjugated antibodies: CD4 (RM4-5; eBioscience), CD25 (37.51; BioLegend), Foxp3 (FJK-16 s; eBioscience), IL-17 (eBio17B7, eBioscience), TNF-α (MP6-XT22; BD Pharmingen), IFN-γ (XMG1.2; eBioscience), IL-4 (11B11; BD Pharmingen), and IL-10 (JES5-16E3; eBioscience). These cells were also intracellularly stained with the following antibodies: TNF-α, IL-17, IFN-γ, IL-10, and Foxp3. Prior to intracellular staining, cells were restimulated for 4 h with 25 ng/ml phorbol myristate acetate (PMA) (Sigma-Aldrich) and 250 ng/ml ionomycin (Sigma-Aldrich) in the presence of GolgiSTOP (BD Pharmingen). Intracellular staining was conducted using an intracellular staining kit (eBioscience) according to the manufacturer’s protocol. Flow cytometric analysis was performed on a FACS_LSR Fortessa (BD Pharmingen).

Lim J.Y., Im K.I., Lee E.S., Kim N., Nam Y.S., Jeon Y.W, & Cho S.G. (2016). Enhanced immunoregulation of mesenchymal stem cells by IL-10-producing type 1 regulatory T cells in collagen-induced arthritis. Scientific Reports, 6, 26851.

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Multiparameter Flow Cytometry Analysis

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FITC-, PerCP-Cy5.5-, APC-, and PE-conjugated monoclonal antibodies for CD3 (17A2), CD4 (RM4-5), CD25 (PC61.5), CD11c (N418), CD8 (53-6.7), Foxp3 (FJK-16s), CTLA4 (UC10-4B9), CD80 (16-10A1), CD86 (GL-1), CD40 (1C10), CD40L (MR1), CD103 (2E7), I-A/I-E (M5/114.15.2), IFN-γ (XMG1.2), IL-6 (MP5-20F3), Granzyme B (GB11-Biolegend), Perforin (eBioOMK-D), and IL-10 (JES5-16E3) antibodies were purchased from eBioscience. Before IFN-γ, IL-6, and IL-10 intracellular staining, cells were stimulated with 50 nM PMA (Sigma-Aldrich, St. Louis, MO, USA), 1 µg/ml ionomycin (Sigma-Aldrich), and 1 µM Brefeldin A (eBioscience) for 4.5 h. After surface staining for 30 min, the cells were suspended in Fixation Buffer (eBioscience), and intracellular cytokine staining was performed as described [18] (link).

Takasato F., Morita R., Schichita T., Sekiya T., Morikawa Y., Kuroda T., Niimi M, & Yoshimura A. (2014). Prevention of Allogeneic Cardiac Graft Rejection by Transfer of Ex Vivo Expanded Antigen-Specific Regulatory T-Cells. PLoS ONE, 9(2), e87722.

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Multiparametric Flow Cytometry of Immune Cells

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Single-cell suspensions of bone marrow, spleen, and lymph nodes were incubated with 1 x ACK solution (for red blood cell lysis) to deplete erythrocytes. Staining was performed with the following antibodies (conjugated with FITC, PE, APC, eFI450, BV786, AF700, PE-Cy7, PerCP Cy5.5, APC-eFI780 or biotin): anti–Siglec-H (551.3D3; Biolegend), anti-mPDCA1 (JF05-1C24.1; Biolegend), anti-CD11c (N418; Biolegend), anti-B220 (RA3-6B2; Biolegend), anti-CCR9 (242503; R&D Systems), anti-FcgR2b (own hybridoma), anti-IL2r (PC61; Biolegend), CD19 (eBio1D3, eBioscience), CD3 (17A2, eBioscience), CD5 (53-7.3, eBioscience), CD4 (GK1.5, eBioscience), CD138 (281-2, BD), CD95 (Jo2, BD), IgD (11-26c.2a, BD), CD80 (16-10A1, eBioscience), PD-L2 (TY25, BD), IL-4 (11B11, BD), IL-6 (MP5-20F3, BD), IL-10 (JES5-16E3, eBioscience), IFN-g (XMG1.2, eBioscience) and Fc-block (2.4G2; own hybridoma). Biotinylated antibodies were detected using streptavidin PerCPCy5.5 or streptavidin PECy7 (Biolegend). Cells were analysed using either Cytoflex (Beckmann Coultier) and FlowJo software (Tree Star).

Szumilas N., Corneth O.B., Lehmann C.H., Schmitt H., Cunz S., Cullen J.G., Chu T., Marosan A., Mócsai A., Benes V., Zehn D., Dudziak D., Hendriks R.W, & Nitschke L. (2021). Siglec-H-Deficient Mice Show Enhanced Type I IFN Responses, but Do Not Develop Autoimmunity After Influenza or LCMV Infections. Frontiers in Immunology, 12, 698420.

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Multiparameter Flow Cytometry Analysis

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We purchased fluorochrome-conjugated mAbs directed against CD4 (RM 4-5), CD8 (53-6.7), Foxp3 (FJK-16 s), IL-10 (JES5-16E3), IFN-γ (XMG1.2) and Ki67 (SolA15) from eBioscience. Flow cytometry was performed on a CytoFLEX flow cytometer (Beckman Coulter), and data analyzed with FlowJo 8 software (Tree-Star).^{25 (link)}

Xu H., Dahiya S., Wang L., Akimova T., Han R., Zhang T., Zhang Y., Qin L., Levine M.H., Hancock W.W, & Levin L.S. (2018). Utility of IL-2 Complexes in Promoting the Survival of Murine Orthotopic Forelimb Vascularized Composite Allografts. Transplantation, 102(1), 70-78.

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Multiparametric Flow Cytometry of Immune Cells

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Monoclonal antibodies used were CD11c (HL3; BD), CD103 (2E7; BioLegend), CD11b (M1/70; eBioscience), CD64 (X54‐5/7.1; BioLegend), MHC class II (M5/114.15.2; eBioscience), CD45 (30‐F11; BioLegend), CD45R (RA3‐6B2; BD), CD8α (53‐6.7; eBioscience), CD197 (4B12; eBioscience), Plet1 (1D4), CD38 (90; eBioscience), CD19 (1D3; eBioscience), CD95 (Jo2; BD), Donkey anti‐rat IgG (Life Technologies), CD4 (GK1.5; BD), Foxp3 (Fjk‐16s; eBioscience), IL‐10 (JES5‐16E3; eBioscience), IFNγ (XMG1.2; BD), and IL‐17A (TC11‐18H10; BD Pharmingen). Zombie Aqua Fixable viability kit (BioLegend) was used for dead cell exclusion.

Karrich J.J., Romera‐Hernández M., Papazian N., Veenbergen S., Cornelissen F., Aparicio‐Domingo P., Stenhouse F.H., Peddie C.D., Hoogenboezem R.M., den Hollander C.W., Gaskell T., Medley T., Boon L., Blackburn C.C., Withers D.R., Samsom J.N, & Cupedo T. (2018). Expression of Plet1 controls interstitial migration of murine small intestinal dendritic cells. European Journal of Immunology, 49(2), 290-301.

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Multiparametric Flow Cytometry Analysis

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The following flow cytometric antibodies were purchased from Biolegend: CD4 (GK1.5), CD8α (53-6.7), CD44 (IM7), IL-17A (TC11-18H10.1), CD3ɛ (17A2), CD45.2 (104), CD45.1 (A20) GITR (YGITR765), ICOS (C398.4A), and PD-1 (RMP1-30), all used at 1:200 dilution. αYFP/GFP (A-21311) was purchased from Life Technologies and used at 1:400 dilution. Antibodies against IFNγ(XMG1.2) used at 1:300 and Ki67 (B56) used at 1:200 dilution were purchased from BD Biosciences. Biotinylated αphospho-S6 was purchased from Cell Signalling Technologies (D57.2.2E) and used at 1:150 dilution. The remaining antibodies for flow cytometry were purchased from eBioscience: CD25 (PC61.5), CD62L (MEL-14), and IL-10 (JES5-16E3) all used at 1:200 dilution; IL-4 (11B11) used at 1:100 dilution; and Foxp3-biotin (FJK-16s). Biotinylated αFoxp3 and αpS6 were used at 1:150 dilution and detected with fluorophore-conjugated streptavidin at 1:750 dilution (S32354 Invitrogen).

Layman A.A., Deng G., O'Leary C.E., Tadros S., Thomas R.M., Dybas J.M., Moser E.K., Wells A.D., Doliba N.M, & Oliver P.M. (2017). Ndfip1 restricts mTORC1 signalling and glycolysis in regulatory T cells to prevent autoinflammatory disease. Nature Communications, 8, 15677.

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Multiparameter Flow Cytometry Protocol

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The following anti-mouse antibodies and conjugates were used in the flow cytometry experiments: Alexa Fluor 647: CD19 (clone 1D3; eBioscience); Alexa Fluor 700: GR-1 (RB6-8C5, eBioscience); APC: CD11b (clone M1/70; eBioscience); IL-10 (JES5-16E3, eBioscience); APC-eFluor780: CD4 (clone GK1.5; eBioscience), CD8 (53–6.7, eBioscience); eFluor-450: Foxp3 (FJK16s, eBioscience); FITC: CD5 (53–7.3, eBioscience); Pacific Blue: CD1d (1B1, Biolegend), CD3 (clone 145-2C11, in house production); PE: IFN-γ (XMG-1.2, eBioscience); PECy5: γδTCR (GL3, eBioscience); PECy7: CD25 (PC61.5, eBioscience), NK1.1 (clone PK136; eBioscience).

Kulkarni U., Herrmenau C., Win S.J., Bauer M, & Kamradt T. (2018). IL-7 treatment augments and prolongs sepsis-induced expansion of IL-10-producing B lymphocytes and myeloid-derived suppressor cells. PLoS ONE, 13(2), e0192304.

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Activation and Phenotyping of T and B Cells

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Cells were stimulated with PMA (10 ng/mL) and ionomycin (500 ng/mL) in the presence of GolgiPlug (1X) and 10% FBS in MEM media for 3-4 hours at 37°C followed by viability using e506 Fixable Viability Dye (eBioscience). Cells were incubated with CD16/CD32 Fc block and extracellular markers were stained with monoclonal antibodies for 20 minutes at 4°C using the following antibodies: CD45 (30-F11), TCRβ (H57-597), CD4 (RM4-5), CD8 (53-6.7), CD19 (1D3). Cells were permeabilized using Foxp3/Transcription Factor Staining kit (eBioscience) and intracellular markers were stained with monoclonal antibodies for 45 minutes at 4°C with FoxP3 (FJK-16S) and IL-10 (JES5-16E3) (All antibodies were from Thermo Fisher Scientific). Cells were resuspended in PBS with 2% NCS and 2 mM EDTA. Samples were collected using Attune NxT Flow Cytometer (Thermo Fisher Scientific) and data analyzed with FlowJo Software (BD Biosciences).

Morse Z.J., Simister R.L., Crowe S.A., Horwitz M.S, & Osborne L.C. (2023). Virus induced dysbiosis promotes type 1 diabetes onset. Frontiers in Immunology, 14, 1096323.

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Multiparametric Flow Cytometry Assay

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Fluorochrome-conjugated, biotinylated antibodies against TCR-β (H57-595), CD4 (RM4-5), CD8 (17A2), CD44 (IM7), CD62L (MEL-14), Foxp3 (FJK-16s), IFN-γ (XMG1.2), IL-4 (11B11), CD98 (RL388), and IL-10 (JES5-16E3) were purchased from Thermo Fisher Scientific. Antibodies against IL-17a (TC11-18H10.1) and Ki67 (B56) were purchased from BD Biosciences. Anti-GzmB (GB11) was purchased from Invitrogen. Antibodies against CC3 (D3E9), p-4EBP1 (236B4), and p-S6 (S235/236) were purchased from CST. Antibody against latency-associated peptide (TW7-16B4) was purchased from BioLegend. Cell surface staining was performed by incubating cells with specific antibodies for 20 min on ice in the presence of 2.4G2 mAb to block FcγR binding. Foxp3, GzmB, IFN-γ, IL-4, IL-17a, IL-10, Ki67, CC3, p-4EBP1, and p-S6 staining was performed using the intracellular transcription factor or cytokine staining kits from Tonbo. Secondary antibodies with fluorochrome conjugation were used for the staining of unconjugated antibodies. To determine cytokine expression, isolated cells were stimulated with 50 ng/ml phorbol o-myristate 13-acetate (Sigma-Aldrich), 1 mM ionomycin (Sigma-Aldrich), and GolgiStop (BD Biosciences) for 4 h before staining. All samples were acquired and analyzed with an LSRII flow cytometer (Becton Dickinson) and FlowJo software (TreeStar).

Do M.H., Wang X., Zhang X., Chou C., Nixon B.G., Capistrano K.J., Peng M., Efeyan A., Sabatini D.M, & Li M.O. (2019). Nutrient mTORC1 signaling underpins regulatory T cell control of immune tolerance. The Journal of Experimental Medicine, 217(1), e20190848.

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Intracellular Cytokine Profiling by Flow Cytometry

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To allow intracellular labelling of cytokines, cell suspensions were stimulated 4 h at 37°C with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma), 1 µg/mL ionomycin (Sigma), and Golgi Stop™ (BD Biosciences). For extracellular labelling, cells were incubated with 200 ng of each antibody. Antibodies targeting extracellular proteins were CD45 (30-F11), PD-1 (RMP1-30) (BD Biosciences), CD3 (17A2), CD4 (GK1.5), CD44 (IM7), CCR9 (9B1), CD11c (N418), CD8a (53-6.7), PD-L2 (24F.10C12), PD-L1 (10F.9G2), MHC-class II (M5/114.5.2), CD80 (16-10A1), CD40 (3/23) (Biolegend), ɣδ-TcR (eBioGL3 (GL-3, GL3), and OVA-dextramer (H-2 Kb) (Immudex) (Table S2).
To allow intracellular labelling, cells were first permeabilized using a FoxP3 staining buffer kit (eBioscience). Antibodies targeting intracellular cytokines were IFNɣ (XMG1.2) (Biolegend), IL-10 (JES5-16E3) (Thermo Fisher Scientific), and IL-17A (TC11-18H10) (BD Biosciences) (Table S2). After intracellular labelling, cells were fixed and stored using FACS Lysing Solution 1X (BD Biosciences). All data were collected using a BD Biosciences FACSCanto II or Lyric and analyzed using FlowJo software.

Boucher E., Plazy C., Richard M.L., Suau A., Mangin I., Cornet M., Aldebert D., Toussaint B, & Hannani D. (2023). Inulin prebiotic reinforces host cancer immunosurveillance via ɣδ T cell activation. Frontiers in Immunology, 14, 1104224.

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