Anti il 2 apc

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-IL-2-APC is a fluorescently-labeled antibody that binds to the interleukin-2 (IL-2) cytokine. It is used for the detection and analysis of IL-2 in flow cytometry applications.

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Anti-IL-2 (14 citations) IL-2-APC (13 citations) Anti-human-IL-2-APC (MQ1-17H12; (2 citations)

7 protocols using anti il 2 apc

Multiparametric Flow Cytometry Analysis

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Cells were harvested and following incubation with Fc block (homemade containing 24G2 supernatant and mouse serum) surface stained with anti‐CD4 APC‐Alexa 780 (eBioscience; clone: RM4‐5), anti‐CD44 PerCP‐Cy5.5 (eBioscience; clone: IM7), CD8 PeCy7 (eBioscience; clone: 53–6.7), and “dump” antibodies: B220 (clone: RA3‐6B2) and MHC II (clone: M5114) both on eFluor‐450 (eBioscience) for 20 min at 4°C. Cells were stained with a fixable viability dye eFluor 506 (eBioscience) as per the manufacturer's recommendations. Cells were fixed with cytofix/cytoperm (BD Bioscience) for 20 min at 4°C and stained in permwash buffer with anticytokine antibodies for 1 h at room temperature (anti‐IFN‐γ PE (clone: XMG1.2;), anti‐TNF Alexa‐Fluor‐488 (clone: MP6‐XT22), anti‐IL‐2 APC (clone: JES6‐5H4) all from eBioscience. Following washing with permwash buffer, samples were acquired on a BD LSR or Fortessa and analyzed using FlowJo (version 10 Treestar). Data are presented as required for MIFlowCyt.

Westerhof L.M., McGuire K., MacLellan L., Flynn A., Gray J.I., Thomas M., Goodyear C.S, & MacLeod M.K. (2019). Multifunctional cytokine production reveals functional superiority of memory CD4 T cells. European Journal of Immunology, 49(11), 2019-2029.

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Ex vivo Stimulation of PBMCs and T Cells

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PBMCs were thawed and stimulated directly ex vivo with or without peptides spanning DENV and ZIKV NS3 and capsid peptide pools (1 μg/ml), or with PMA/ionomycin as a positive control for 5 h at 37°C in the presence of Brefeldin A (10 μg/ml, Sigma-Aldrich). Anti CD107a FITC (clone H4A3) antibody was added to the cells at the beginning of the stimulation to assess their degranulation capacity. Similarly, T cells lines were stimulated with or without the peptide of interest (1 μg/ml) with in some cases addition of anti CD107a FITC antibody at the beginning of the stimulation. After stimulation cells were stained with the yellow LIVE/DEAD fixable dead cell stain kit (Invitrogen) and surface stained with anti CD8 PECY7 (clone RPA-T8) and anti CD4 BV 650 (clone RPA-T4) antibodies, then fixed, and permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen). Cells were then stained with anti IFN-γ PE (R&D, clone 25723), anti IL-2 APC (eBioscience, clone MQ1-17H12), and/or anti TNF-α FITC antibodies (clone 6401.1111) and analyzed on a BD LSR II. Antibodies were purchased from BD unless otherwise stated.

Lim M.Q., Kumaran E.A., Tan H.C., Lye D.C., Leo Y.S., Ooi E.E., MacAry P.A., Bertoletti A, & Rivino L. (2018). Cross-Reactivity and Anti-viral Function of Dengue Capsid and NS3-Specific Memory T Cells Toward Zika Virus. Frontiers in Immunology, 9, 2225.

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Intracellular Cytokine Staining of PBMC

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Isolated 10⁶ PBMC were stimulated with phorbol myristate acetate (PMA) (25 ng/ml) and ionomycin (1 μg/ml) for 6 h at 37°C in RPMI‐1640 medium containing 10% FCS and 10 μg/ml brefeldin A. Immediately after stimulation, the cells were stained with anti‐CD3‐APC/Cy7 (BioLegend; clone HIT3a), anti‐CD4‐PerCP (BioLegend; clone RPA‐T4), anti‐CD8‐PE/Cy7 (eBioscience; clone RPA‐T8) and anti‐CD45RO‐Pacific Blue (BioLegend; clone UCHL1). Subsequently, cells were fixed and permeabilized using Cytofix/Cytoperm (BD), washed and stained for intracellular IL‐2 in Perm buffer with anti‐IL‐2‐APC [eBioscience; clone MQ1–17H12, in parallel with interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α], assessed cytometrically with a fluorescence activated cell sorter (FACS)Canto II cytometer and analysed using FlowJo software (TreeStar, Inc.).

Costa N., Marques O., Godinho S.I., Carvalho C., Leal B., Figueiredo A.M., Vasconcelos C., Marinho A., Moraes‐Fontes M.F., Gomes da Costa A., Ponte C., Campanilho‐Marques R., Cóias T., Martins A.R., Viana J.F., Lima M., Martins B, & Fesel C. (2017). Two separate effects contribute to regulatory T cell defect in systemic lupus erythematosus patients and their unaffected relatives. Clinical and Experimental Immunology, 189(3), 318-330.

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Cytokine Production Assay in Immune Cells

Cited 1 time

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Splenocytes or peripheral blood mononuclear cells (PBMC) were stimulated with peptide at 2 µg/mL; ionomycin and phorbol myristate acetate (PMA) at 2.0 mg/mL and 0.5 mg/mL, respectively, as positive assay controls; tissue culture medium with 1% DMSO was used as a negative control and processed as previously described [17 (link)]. The following mAb reagents were used: anti-CD107a phycoerythrin (PE)-conjugated mAb, anti-CD3 PerCP-eFluor710, anti-CD8a eFluor 450, anti-IFN-γ PE-Cy7, anti-IL-2 APC, and anti-tumor necrosis factor (TNF)-α fluorescein isothiocyanate (FITC) (all from eBioscience, San Diego, CA, USA) and anti-CD4 allophycocyanin (APC)/Cy7 (BioLegend, San Diego, CA, USA). Fixed cells were acquired on an LSRII flow cytometer (Becton Dickinson, Wokingham, UK). All mice were assayed in triplicates.

Moyo N., Wee E.G., Korber B., Bahl K., Falcone S., Himansu S., Wong A.L., Dey A.K., Feinberg M, & Hanke T. (2020). Tetravalent Immunogen Assembled from Conserved Regions of HIV-1 and Delivered as mRNA Demonstrates Potent Preclinical T-Cell Immunogenicity and Breadth. Vaccines, 8(3), 360.

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Stimulation and Analysis of Mouse T Cells

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Mouse cell suspensions were stimulated with 50 μg/mL of TT or HEL peptide in the presence of BD GolgiPlug™ (BD Biosciences) for 2 h (37 °C). Cells were fixed and stained using the BD Cytofix/Cytoperm™ Kit (BD Biosciences). Surface and intracellular staining (ICS) were performed according to the manufacturer’s instructions, with anti-CD4-FITC, anti-CD3-APC-eFluor^®780, anti-CD19-PECy7, anti-TNF-α-PerCP-e710, anti-IL-2-APC and anti-IFNγ-EF450 from eBioscience and anti-CD8a-PE from BD Biosciences.

Laubreton D., Bay S., Sedlik C., Artaud C., Ganneau C., Dériaud E., Viel S., Puaux A.L., Amigorena S., Gérard C., Lo-Man R, & Leclerc C. (2016). The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity. Cancer Immunology, Immunotherapy, 65, 315-325.

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Immunization and Analysis of Arthritis in Mice

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Flt3L^−/−, WT and Batf3^−/− mice were immunized as previously described [23 (link)] and were inspected three times a week for signs of arthritis by two independent observers. All mice were sacrificed on day 43 after CIA induction. Blood, LNs and paws were harvested for analysis. Single-cell suspensions were obtained and after erythrocyte lysis (red blood cell lysis buffer, 2 min at RT; Sigma), cells were stained with the indicated fluorochrome-conjugated antibodies for surface markers and intracellular cytokines. LN cells were stained using the following markers: anti-TNF (APC, eBioscience), anti-IL-2 (APC, eBioscience), anti-IL-17 (Alexa 488, eBioscience), anti-IFNγ (PercP Cy5.5, eBioscience), anti-IL10 (PE, eBioscience), anti-GM-CSF (PE, eBioscience), anti-B220 (PerCP, eBioscience), anti-CD19 (Alexa 700, eBioscience), anti-GL7 (biotin, eBioscience), anti-IgD (PE, BD Pharmingen), anti-CD38 (FITC, eBioscience), anti-CD95 (APC, eBioscience) and streptavidin (PE-Cy7, eBioscience). Synovial cells were isolated and stained using antibodies against CD11c (PE-Cy7, eBioscience), MHCII (APC-Cy7, eBioscience), CD11b (Alexa 700) and CD103 (FITC, eBioscience). Serum levels of antibodies against chicken collagen type II (cCII) were measured by ELISA. Further details are described in supplementary methods.

Ramos M.I., Garcia S., Helder B., Aarrass S., Reedquist K.A., Jacobsen S.E., Tak P.P, & Lebre M.C. (2020). cDC1 are required for the initiation of collagen-induced arthritis. Journal of Translational Autoimmunity, 3, 100066.

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Antigen-Specific T Cell Activation

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Antigen specific T cells were cocultured with T2 cells pulsed with the cognate peptide versus a control peptide for 2 hours. Brefeldin A (eBioscience) was added to the coculture for another 4 hours. The cells were then fixed in 2% paraformaldehyde, permeabilized, and stained with anti-CD3 and anti-CD8 (BD Biosciences) along with anti-IFN-γ-PE-Cy7 and anti-IL-2-APC (eBioscience). Cytokine staining was assessed on CD3+CD8+ gated cells.

Chandran S.S., Paria B.C., Srivastava A.K., Rothermel L.D., Stephens D.J., Dudley M.E., Somerville R., Wunderlich J.R., Sherry R.M., Yang J.C., Rosenberg S.A, & Kammula U.S. (2014). Persistence of CTL clones targeting melanocyte differentiation antigens was insufficient to mediate significant melanoma regression in humans. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(3), 534-543.

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