α il 4

Manufactured by Thermo Fisher Scientific

Sourced in United States

α-IL-4 is a lab equipment product that measures the concentration of interleukin-4 (IL-4) in biological samples. It is used as a tool for research and scientific investigation.

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α-IL-4 antibody (1 citations)

8 protocols using α il 4

Differentiation of Naive T Cells

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Naive T cells were isolated by sorting CD4⁺CD25⁻CD62L^highCD44^low cells from spleens and lymph nodes, differentiated under several Th cell conditions, and analyzed as described (Yang et al., 2008 (link)). The naïve T cells (5 × 10⁵ cells/well) were stimulated with the plate-bound α-CD3 (1 μg/ml) and the soluble α-CD28 (1 μg/ml). For Th0 differentiation, the cells were treated with 5 μg/ml α-IFN-γ (XMG1.2; BioXCell), 5 μg/ml α-IL-4 (11B11; BioXCell) and 30 U/ml IL-2. For Th1 differentiation, the cells were treated with 10 ng/ml mIL-12 (Peprotech), 5 μg/ml α-IL-4 and 30 U/ml IL-2. For Th2 differentiation, the cells were treated with 10 ng/ml mIL-4 (Peprotech), 5 μg/ml α-IFN-γ. For iTreg differentiation, the cells were treated with 1 ng/ml TGF-β1 (Peprotech), 5 μg/ml α-IFN-γ, 5 μg/ml α-IL-4 and 30 U/ml IL-2. For Th17 differentiation, the cells were treated with 0.5 ng/ml TGF-β1, 10 ng/ml IL-6 (Peprotech), 5 μg/ml α-IFN-γ, and 5 μg/ml α-IL-4. When indicated, 10 ng/ml IL-23 and 10 ng/ml IL-1β (Peprotech) were used for optimal Th17 cell differentiation.

Ichiyama K., Chen T., Wang X., Yan X., Kim B.S., Tanaka S., Ndiaye-Lobry D., Deng Y., Zou Y., Zheng P., Tian Q., Aifantis I., Wei L, & Dong C. (2015). The methylcytosine dioxygenase Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells. Immunity, 42(4), 613-626.

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In vitro Naive CD4+ T cell Polarization

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Naïve CD4⁺ T cells were sorted from the spleens and lymph nodes with the MACS CD4⁺CD62L⁺ T Cell Isolation (30–093-227) Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured under neutral (TH0) conditions in supplemented IMDM medium in the presence of activating antibodies (5 μg/ml plate-coated anti-CD3 and 1 μg/ml soluble anti-CD28) and iTreg polarizing cytokines: TGF-β [5 ng/ml], human IL-2 [20 ng/ml], αIL-4 [2 μg/ml], αIFN-γ [2 μg/ml] and αIL-12 [2 μg/ml].
Recombinant proteins (recombinant human IL-2 and TGF-β) and blocking antibodies (anti-mouse IL-4, anti-mouse IFN-γ, anti-mouse IL-12) for in vitro cell differentiation were purchased from eBioscience (San Diego, California,USA).

Thuille N., Siegmund K., Klepsch V., Schörgenhuber J., Danklmaier S., Leitges M, & Baier G. (2019). Loss-of-function phenotype of a PKCθT219A knockin mouse strain. Cell Communication and Signaling : CCS, 17, 141.

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Macrophage and Microglia Profiling in Hippocampus

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Flow cytometry was used to determine macrophage and microglia populations in single cell preparations of the hippocampus prepared in complete media; IMDM (GIBCO/Invitrogen; Carlsbad, CA), 10% FCS, and P/S on ice. Samples were stained with an antibody mix (MACS buffer +1% inactivated Rat serum, 1% α-FcγII/III (clone 2.4G2), α-F4/80 (clone GK1.5; BD Pharmingen^TM, FITC), and α-Iba1 (clone GR106078-1; Abcam®, FITC) for 30 min on ice, and then fixed in 2% paraformaldehyde before permeabilization (0.5 g saponin, 0.055 g CaCl2, 0.0625 g MgSO4, 0.25 g NaN3, 0.5 g BSA, 10 mM HEPES in final volume of 500 ml of 1X PBS) for 1 hour at 4 °C. Intracellular staining of samples was done with an antibody mix (MACS buffer +2% inactivated Rat serum, 2% α-FcγII/III (clone 2.4G2), α-IL-1α (clone ALF-161; BioLegend®, PE); α-IL-6 (Lot 99523; BP Pharmingen^TM, PE); α-iNOS2 (clone Sc651; Santa Cruz Biotechnology®), α-MHCII (clone M5/114.15.2; eBioscience®, AlexaFluor 700), α-IL-4 (clone 11B11; eBioscience®, APC); α-IL-13 (clone eBio13A; eBioscience®, PE Cy7); and α-Arginase1 (clone H1010; Santa Cruz Biotechnology®) for 30 min on ice and read by a Becton Dickinson FACS FORTESSA machine (BD San Diego, CA). Data was analyzed by FlowJo© Treestar (Ashland, OR) and graphed with GraphPad Prism® software. Unless otherwise stated, antibodies were from BD Pharmingen^TM.

Brombacher T.M., De Gouveia K.S., Cruywagen L., Makena N., Booley F., Tamgue O, & Brombacher F. (2018). Nippostrongylus brasiliensis infection leads to impaired reference memory and myeloid cell interference. Scientific Reports, 8, 2958.

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Murine T Cell Activation Assay

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Purified α-CD3 (145-2C11), purified α-CD28, α-CD25 (3C7), Biotin α-CD25, fluorescein isothiocyanate (FITC)-α-CD4, phycoerythrin (PE)-α-CD25, unconjugated, (allophycocyanin) APC, Alexafluor-647 or PE conjugated α-CD44, APC, Alexafluor 647, PE or PE-Cy7 conjugated IL-17-A, Foxp3 antibodies, IL-6 blocking antibodies, α-IL-4 and IFN-γ antibodies, were all purchased from eBiosciences (San Diego, CA). Mouse CD4 isolation kit II was purchased from Miltenyi Biotec (Auburn, CA). EasySep isolation kits were purchased from Stem Cell technologies (Canada). Human IL-2 purchased from NCI, was a kind gift from Mike Lenardo lab (NIAID, NIH). Recombinant mouse IL-6 was purchased from BioBasic Inc (Amherst, NY), and human TGF-β was purchased from R&D Systems (Minneapolis, MN). Mouse cells were cultured in complete RPMI-1640 (Bio-Whittaker) supplemented with 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 5 mM Glutamax (Invitrogen) and 50 μM β-mercaptoethanol.

Bhaskaran N., Cohen S., Zhang Y., Weinberg A, & Pandiyan P. (2015). TLR-2 Signaling Promotes IL-17A Production in CD4+CD25+Foxp3+ Regulatory Cells during Oropharyngeal Candidiasis. Pathogens, 4(1), 90-110.

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Multiparametric Flow Cytometry Analysis

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Cells were labeled with fluorescence antibodies for mouse α-CD4, α-CD8, α-INF-γ, α-IL-2, α-IL-10, α-IL-4, α-IL-17A, α-CD44, and α-CD62L (eBioscience, CA). To assess immune responses after transplantation, total splenocytes or isolated CD4⁺ T cells were seeded on 48 well plates and stimulated in complete media for 4 hrs at 37°C with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma-Aldrich), Ionomycin (500ng/ml; Sigma-Aldrich) and Brefeldin A (eBioscience). Thereafter, cells were fixed, permeabilized and stained with respective antibodies at a concentration of 1–5 μg per 10⁶ cells. Flow cytometry measurements were performed on a FACS Canto II (BD Bioscience, CA, USA) and data were analyzed using FlowJo (FlowJo Software, OR, USA).

Krenzien F., Quante M., Heinbokel T., Seyda M., Minami K., Uehara H., Biefer H.R., Schuitenmaker J.M., Gabardi S., Splith K., Schmelzle M., Petrides A.K., Azuma H., Pratschke J., Li X.C., ElKhal A, & Tullius S.G. (2016). Age-dependent metabolic and immunosuppressive effects of Tacrolimus. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 17(5), 1242-1254.

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Generation and Characterization of Gimap5 Knockout Mice

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All experiments were performed according to US National Institutes of Health guidelines and were approved by the IACUC of The Cincinnati Children’s Hospital. C57BL/6J mice were obtained from Jackson. Deletion of Gimap5 in Gimap5^flox/floxCd4^cre-ert2 mice was induced by the administration of tamoxifen chow (40mg/kg body weight; Harlan Laboratories Teklad Diets). Gimap5^sph/sph mice were generated as previously described (6) and bred in-house in the vivarium of Cincinnati Children’s Hospital. All mice were maintained under specific pathogen-free conditions.
All antibodies used for flow cytometry were purchased from eBioscience or Biolegend unless otherwise noted. Purified α-mouse-CD3 (17A2) and α-mouse-CD28 (37.51) antibodies (Biolegend) were used for mouse T cell activation. A fixable viability stain was purchased from eBioscience. 7-AAD was purchased from BD. PMA, ionomycin, and Brefeldin A were obtained from Sigma. αIFNγ (XMG1.2) and mIL-23 were obtained from eBioscience. TGFβ1, mIL-4, and hIL-2 were purchased from Miltenyi Biotech. αIL-4, IL-6, and mIL-1β, mIL-12, and TGFβ3 were obtained from PeproTech.

Patterson A.R., Bolcas P., Lampe K., Cantrell R., Ruff B., Lewkowich I., Hogan S.P., Janssen E.M., Bleesing J., Khurana Hershey G.K, & Hoebe K. (2018). Loss of Gimap5 promotes pathogenic CD4+ T cell development and allergic airway disease: Gimap5-deficiency predisposes CD4+ T cells to Th17 polarization. The Journal of allergy and clinical immunology, 143(1), 245-257.e6.

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In vitro Treg and T cell differentiation

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Sorted cells were re-suspended in complete clicks media (Irvine Scientific) along with 10% FBS, 1% Pen/Strep, 1% L-glut (2mM), 55μM βMe. 48 well flat bottom plates were coated with plate bound αCD3 (2μg/ml, Clone 145–2C11) and αCD28 (2ug/ml, Clone 37.51) in 1X PBS overnight. For proliferation and suppression assays, cells were labeled with cell-trace violet (Life Technologies) as indicated. The in vitro differentiation assay conditions used for 3 days were: tTreg and Th0- h.rIL-2 (RnD systems) 100IU/ml; iTreg- IL-2 100 IU/ml, h rTGF-β 10ng/ml (Peprotech), α-IL4 and α-IFNγ 10μg/ml; Th1- h.r.IL-2 100IU/ml, mouse IL-12 5ng/ml (Peprotech) and α-IL4 10ug/ml. For differentiated cells analyzed at day 5, cells were split 1:2 on day 3 and rested with IL-2 100IU/ml for 48 hours before analysis as described previously (10 (link)). For suppression assays, Treg cells at day 5 were cocultured with labeled CD45.1⁺ total T cells in the presence of soluble αCD3 at 0.25ug/ml and irradiated APCs (5×10⁴ cells) at the indicated Treg:T cell ratios for 3 days and proliferation of T responders was analyzed on day 8 (19 (link)).

Priyadharshini B., Loschi M., Newton R.H., Zhang J.W., Finn K.K., Gerriets V.A., Huynh A., Rathmell J.C., Blazar B.R, & Turka L.A. (2018). TGF-β and PI3K Signals Modulate Distinct Metabolism of Treg Subsets. Journal of immunology (Baltimore, Md. : 1950), 201(8), 2215-2219.

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Activation of Primary CD4+ T Cells

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Primary CD4 + T cells were isolated from buffy coats from healthy donors by Ficoll gradient followed by density-based negative selection of CD4+ T cells with RosetteSep kit (StemCells Technologies). Twenty four hours after isolation primary CD4+ T cells were cultured in the presence of 10 ng/ml TGF-β (Sigma-Aldrich), 1 µg/ml α-IL-4 (PeproTech) and αCD3/CD28 dynabeads (Life Technologies) at the cell:bead ratio 1:1 for 3 days. αCD3/CD28 dynabeads were removed, cells washed and cultured for 4 days in growth media supplemented with 30 IU/ml rIL-2 (Roche) Then cells were washed and subjected to spininfection (90 min, 1200 g) and incubated over-night. Next day cells were washed and re-suspended in growth media supplemented with 30 IU/ml rIL-2 and Saquinavir Mesylate (5 µM). Seven days post-infection cells were treated with BRD-K80443127 in increasing concentrations or with PMA/Ionomycin in the presence of Raltegravir (30 µM). After 24 hours of stimulation cells were collected and subjected to the luciferase assay, RLU was normalized to the total protein content.

Marian C.A., Stoszko M., Wang L., Leighty M.W., de Crignis E., Maschinot C.A., Gatchalian J., Carter B.C., Chowdhury B., Hargreaves D.C., Duvall J.R., Crabtree G.R., Mahmoudi T, & Dykhuizen E.C. (2018). Small Molecule Targeting of Specific BAF (mSWI/SNF) complexes for HIV Latency Reversal. Cell chemical biology, 25(12), 1443-1455.e14.

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