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Human il 2

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Human IL-2 is a recombinant protein that represents the full-length sequence of the human interleukin-2 (IL-2) cytokine. IL-2 is a key regulator of T-cell and natural killer cell activities, and is essential for the proliferation and differentiation of these cells.

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Lab products found in correlation

Human il 4, by BioLegend (2 mentions) Human gm csf, by BioLegend (2 mentions) Human ifn γ, by BioLegend (2 mentions) Fluorophore conjugated anti cd107a, by BioLegend (2 mentions) 96 well high binding flat bottom plates, by Thermo Fisher Scientific (2 mentions) Golgistop, by BD (2 mentions) Golgiplug, by BD (2 mentions) Retronectin coated plate, by Takara Bio (2 mentions) Aim 5 media, by Thermo Fisher Scientific (2 mentions) Lympholyte h density separation, by Cedarlane (2 mentions) Anti cd3 antibody okt3, by BioLegend (2 mentions) Human ab serum, by Merck Group (2 mentions) Cd8 microbead, by Miltenyi Biotec (1 mentions) Hyaluronidase type 5, by Merck Group (1 mentions) Doxycycline hyclate, by Merck Group (1 mentions) Dnase type 4, by Merck Group (1 mentions) Murine il 2, by BioLegend (1 mentions) Percoll density gradient media, by Merck Group (1 mentions) Murine ifn γ, by Abcam (1 mentions) Anti pd 1, by BioXCell (1 mentions)

Spelling variants of this product

Recombinant human IL-2 (44 citations) Human IL-2 ELISA kit (8 citations) ELISA MAX™ Deluxe Set Human IL-2 (7 citations) Human IL-2 ELISA MAX Deluxe (4 citations) LEGEND MAX™ Human IL-2 ELISA Kit (3 citations) Human IL-2 ELISA MAX Standard Kit (2 citations) Recombinant human IL-2 and IL-15 (2 citations) PE-anti-human IL-2 (2 citations) Human IL-2 ELISA MAXTM Delux (2 citations) Human IL-2 ELISA Max Deluxe kit (2 citations)

14 protocols using human il 2

1

Isolation and Differentiation of DCs and CD8+ T Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque density gradient centrifugation and cultured in RPMI 1640 medium containing 10% FCS for 2 hours. DCs, which were generated from the adherent fraction of PBMCs [38 (link)], were cultured for 5 days in RPMI 1640 medium containing 10% FCS, 20 ng/mL human GM-CSF, and 10 ng/mL human IL-4 (Biolegend). Culture medium and cytokines were refreshed in every other day. On the day 5, the medium was replaced by rituximab-treated conditioned medium or control medium and cell culture was continued for 24 hours. Autologous CD8+ T lymphocytes were magnetically isolated using CD8 MicroBeads (Miltenyi Biotech, Germany) to obtain purity ≥95% CD8+ T cells. The isolated T lymphocytes were cultured in RPMI 1640 medium containing 10% FCS and 10 ng/mL human IL-2 (Biolegend) and the medium was replaced every other day [37 (link)].
Zhao T., Ren H., Wang X., Liu P., Yan F., Jiang W., Li Y., Li J., Gribben J.G., Jia L, & Hao J. (2015). Rituximab-induced HMGB1 release is associated with inhibition of STAT3 activity in human diffuse large B-cell lymphoma. Oncotarget, 6(29), 27816-27831.
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2

Quantification of Secreted Proteins and Antibody Binding

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To determine the concentration of secreted proteins after stimulation, human IFN-γ (#430101; BioLegend) and human IL-2 (#431801; BioLegend) detection kits were used. To quantify anti-CD45 and anti-SLAMF6 antibody binding, polystyrene high binding microplates (Corning) were coated with immobilized CD45 (#14197-H08H; SinoBiological) or SLAMF6 (#11945-H08H; SinoBiological) recombinant ectodomain proteins, respectively. To quantify anti-CD3 and/or anti-SLAMF6 antibody binding to CD3, immobilized SLAMF6 KO Jurkat T cell lysates were used as antigen bait. Primary antibody binding was detected using a secondary HRP goat antibody recognizing human Fc (#SSA001-200; Sino-Biologic).
Gartshteyn Y., Askanase A.D., Song R., Bukhari S., Dragovich M., Adam K, & Mor A. (2022). SLAMF6 compartmentalization enhances T cell functions. Life Science Alliance, 6(2), e202201533.
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3

Cytokine and Antibody Modulation in Vivo

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The following cytokines were used: Human IL-2 (BioLegend 589104), murine IL-2 (BioLegend 575406), murine TGFβ1 (BioLegend 763104), murine IFNγ (Abcam #Ab9922), human IFNγ (PeproTech #300–02). In vivo experiments were performed with the following mAbs: inVivoMAb mouse anti-PD1 (BioXcell RMPI-14 clone), inVivoMAb rat IgG2a, isotype control (BioXcell 2A3 clone) and CD8α depletion antibody (BioXcell, 2.43 clone). Other reagents included Doxycycline hyclate (Sigma-Aldrich #D9891), collagenase type IV (Sigma-Aldrich #C5138), DNAse type IV (Sigma-Aldrich #D5205), Hyaluronidase Type V (Sigma-Aldrich #H6254), ACK lysis buffer (Life Technologies #A1049201), Percoll density gradient media (Sigma-Aldrich #P1644) and TGFβ receptor I inhibitor Galunisertib, LY2157299 (Selleck #S2230).
Bagati A., Kumar S., Jiang P., Pyrdol J., Zou A., Godicelj A., Mathewson N.D., Cartwright A.N., Cejas P., Brown M., Giobbie-Hurder A., Dillon D., Agudo J., Mittendorf E.A., Liu X.S, & Wucherpfennig K.W. (2020). Integrin αvβ6 – TGFβ – SOX4 Pathway Drives Immune Evasion in Triple-negative Breast Cancer. Cancer cell, 39(1), 54-67.e9.
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4

Multiparameter Analysis of Immune Cells

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Anti-human PD-L1/CD274, -PE and –APC (Clone# 29E.2A3, catalog#329745, 329706, 329708), anti-FLAG antibody (Clone# L5, catalog#637304), Alexa Fluor 555 goat anti-rat IgG antibody (Catalog#405420), Alexa Fluor 467 goat anti-rat IgG antibody(Catalog#405416), human IFNγ (Catalog#570208), human IFNα (Catalog#592704), human IL-2 (Catalog#589104), and Zombie UV dye (Catalog#77474) were purchased from Biolegend (San Diego, CA, USA). Human IFNβ (Catalog#8499-1F) was purchased from R&D Systems (Minneapolis, MN 55413). Heat Inactivated human AB serum was purchased from Innovative Research, Inc (Catalog# IPLA-SERAB-27146, MI 48377, USA). Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation was purchased from ThermoFisher Scientific (Catalog# 11131D, Waltham, MA, USA). The CF33-hNIS-Δ (CF33-hNIS-ΔF14.5L) virus [24 (link), 25 (link)], CF33-GFP [ref.26 (link)], and the CF33-hNIS-antiPDL1 virus [23 (link)] were generated in our lab as previously described. Anti-sodium/Iodide Symporter (hNIS) monoclonal antibody was purchased from EMD Millipore Corp (Catalog# MAB3564, Billerica, MA, USA). Goat anti-rabbit IgG (H+L) Alexa Fluor 555 antibody (Catalog# A21434) and Goat anti-mouse Alexa Fluor 488 antibody (Catalog# A11029) were purchased from Invitrogen Corporation (Carlsbad, CA, USA).
Zhang Z., Yang A., Chaurasiya S., Park A.K., Lu J., Kim S.I., Warner S.G., Yuan Y.C., Liu Z., Han H., Von Hoff D., Fong Y, & Woo Y. (2021). CF33-hNIS-antiPDL1 Virus Primes Pancreatic Ductal Adenocarcinoma for Enhanced anti-PD-L1 Therapy. Cancer gene therapy, 29(6), 722-733.
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5

Generation of Dendritic Cells from PBMCs

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Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque (Solarbio Co., China) density gradient centrifugation and cultured in RPMI 1640 medium containing 10% FBS for 2 hours. DCs were generated from the adherent fraction of PBMCs [48 (link)]. The adherent cells were cultured for 6 days in RPMI 1640 medium containing 10% FBS, 20 ng/mL human GM-CSF, and 10 ng/mL human IL-4 (Biolegend, USA). Culture medium and cytokines were refreshed every other day. On the day 6, the medium was replaced by the Gem or/and PX-478-treated conditioned medium or control medium and cell culture was continued for 24 hours. Autologous CD3+ T lymphocytes were magnetically isolated using CD3 MicroBeads (Miltenyi Biotech, Germany) to obtain purity ≥95% CD3+ T cells. The isolated T lymphocytes were cultured in RPMI 1640 medium containing 10% FBS and 10 ng/mL human IL-2 (Biolegend, USA), and the medium was replaced every other day.
Zhao T., Ren H., Jia L., Chen J., Xin W., Yan F., Li J., Wang X., Gao S., Qian D., Huang C, & Hao J. (2014). Inhibition of HIF-1α by PX-478 enhances the anti-tumor effect of gemcitabine by inducing immunogenic cell death in pancreatic ductal adenocarcinoma. Oncotarget, 6(4), 2250-2262.
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6

Immunosuppressive Effects of MSCs

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MSC function was assessed by measuring their ability to inhibit the proliferation of, and cytokine production by, activated T cells. In brief, MSCs (2×104) were irradiated at 30-Gy to inhibit proliferation, then were painted for 30 min at 37°C in the presence or absence of activated CFH (10 μg/ml). After washing, these cells were incubated with 30% NHS in GVB++ buffer for 30 min at 37°C, washed, and incubated with different numbers of PBMCs (MSC: PBMC ratio 1:10, 1:20 and 1:40) in the presence of anti-CD3/28 Dynabeads (2 μl, ThermoFisher, Waltham, MA) and human IL-2 (30 U/ml; Biolegend, CA). After 48 h, BrdU (10 μM) was added to the co-cultures and culture supernatants were collected 72 h later and IFN-r levels measured by conventional ELISA (Biolegend, CA), while the cells were fixed and proliferation of the activated T cells assessed by measuring BrdU incorporation using a BrdU ELISA kit (Roche, Chicago, IN).
Li Y., Qiu W., Zhang L., Fung J, & Lin F. (2016). Painting factor H onto mesenchymal stem cells protects the cells from complement- and neutrophil-mediated damage. Biomaterials, 102, 209-219.
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7

Activation and Differentiation of Immune Cells

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The following conditions were used for B1 cell and CD4+ T cell stimulation. Enriched B1 cells were activated using 2.5 mg/mL LPS (ultrapure, Invivogen) in the presence of mouse IL-4, mouse IL-5 and mouse IL-6 (each 100 ng/mL; Biolegend) for 24 h at 37 C. Naı ¨ve CD4+ T cells were activated with ionomycin (1 mM; Life technologies) and phorbol 12-myristate 13-acetate (PMA), (10 ng/mL; Sigma Aldrich) or by incubation with surface-bound antibodies against CD3ε and CD28 (Biolegend). For surface coating of 96-well plates, 100 mL antibody mix containing 1 mg of each antibody in PBS was used per well and incubated for 2-3 h at 37 C followed by a washing step with 200 mL PBS.
Naı ¨ve CD4+ T cells were differentiated into FoxP3-expressing T cells by cultivation in anti-CD3ε/CD28-coated 96-well plates in the presence of human IL-2 (20 ng/mL; Biolegend), mouse IL-7 (100 ng/mL; Biolegend), mouse IL-15 (100 ng/mL; Biolegend), human TGF-beta1 (5 ng/mL; Biolegend) and retinoic acid (10 nM; Sigma-Aldrich) at 37 C for at least 48 h. FoxP3-expression was confirmed by flow cytometry after intracellular staining using the BD Cytofix/Cytoperm Kit and anti-FoxP3 antibody (clone MF-14; Biolegend).
Engels R., Falk L., Albanese M., Keppler O.T, & Sewald X. (2022). LFA1 and ICAM1 are critical for fusion and spread of murine leukemia virus in vivo. Cell reports, 38(3).
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8

Naïve CD4+ T Cell Activation Assay

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Purified naïve CD4+ T cells were cultured in serum‐free ImmunoCult‐XF T Cell Exp Medium (StemCell, Vancouver, BC, Canada), stimulated by ImmunoCult™ Human CD3/CD28 T Cell Activator (Squirrel‐Bio, Guangzhou, China) and human IL‐2 (BioLegend), and incubated in the presence of CMs from cells overexpressing the four wild‐type or mutant genes for 48 h. Next, they were stained with CD4‐APC (BioLegend), T‐bet‐PerCP/Cyanine5.5 (BioLegend) and GATA‐3 Alexa Fluor® 488 (BioLegend) for flow cytometry analysis.
Miao L., Wei X., Zhao Q., Qi J., Ren C., Wu Q., Wei D., Liu J., Wang F, & Xu R. (2020). p.P476S mutation of RBPJL inhibits the efficacy of anti‐PD‐1 therapy in oesophageal squamous cell carcinoma by blunting T‐cell responses. Clinical & Translational Immunology, 9(9), e1172.
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9

NK Cell Responsiveness Assay

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To analyze the responsiveness of NK cells ex vivo, 96-well high-binding flat-bottom plates (Thermo Fisher) were coated overnight with PBS plus anti-NKp46 or control Ig at 5 ug/ml. Plates were washed three times with PBS before stimulation. Cells were cultured in the coated plates for 5 hr in the presence of Golgi-Stop and Golgi-Plug (1:1000 each) (BD Biosciences), 1000 U/ml human IL-2, and fluorophore-conjugated anti-CD107a (0.5 ug/ml) (Biolegend). After stimulation, cells were stained for extracellular markers to identify NK cells and then subjected to intracellular staining for IFN-γ, followed by flow cytometry analysis.
Thompson T.W., Jackson B.T., Li P.J., Wang J., Kim A.B., Huang K.T., Zhang L, & Raulet D.H. (2018). Tumor-derived CSF-1 induces the NKG2D ligand RAE-1δ on tumor-infiltrating macrophages. eLife, 7, e32919.
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10

Assessing NK Cell Activation Ex Vivo

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To analyze the responsiveness of NK cells ex vivo, 96-well high-binding flat-bottom plates (Thermo Fisher) were coated overnight with PBS plus the indicated antibody against NK activating receptors. Plate-bound antibodies were coated at the following concentrations: anti-NK1.1: 50 μg/ml; anti-NKG2D: 5 μg/ml; anti-NKp46: 5 μg/ml. Plates were washed three times with PBS before stimulation. Single-cell suspensions were generated from the indicated tissue and cultured in the coated plates for 5 hr in the presence of Golgi-Stop and Golgi-Plug (1:1000 each) (BD Biosciences), 1000 U/ml human IL-2, and fluorophore-conjugated anti-CD107a (0.5 μg/ml) (Biolegend). After stimulation, cells were stained for extracellular markers to identify NK cells and then subjected to intracellular staining for IFN-γ, followed by flow cytometry analysis.
Thompson T.W., Kim A.B., Li P.J., Wang J., Jackson B.T., Huang K.T., Zhang L, & Raulet D.H. (2017). Endothelial cells express NKG2D ligands and desensitize antitumor NK responses. eLife, 6, e30881.
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