Il 4 pe

Manufactured by Thermo Fisher Scientific

Sourced in United States

IL-4-PE is a fluorescent-labeled antibody that can be used to detect and quantify interleukin-4 (IL-4) in biological samples. IL-4 is a cytokine that plays a key role in the immune response and is involved in various physiological and pathological processes.

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Anti-IL-4-PE PE‐labeled anti‐mouse IL‐4 antibody IL-4-PE-Cyanine7 PE-conjugated anti-IL-4 PE-Cy7-conjugated IL-4 Pe-Cy7-conjugated anti-IL-4

23 protocols using il 4 pe

Plasmid-based PTEN Gene Therapy

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The three plasmid systems, including target gene plasmid (pAAV-CAG-mCherry), capsid plasmid (pAAV-6, pAAV-1, pAAV-7m8, pAAV-7, pAAV-8, pAAV-10) and helper plasmid (pHelper), were all purchased from Fenghui Biotechnology (Hunan, China). The complementary DNA (cDNA) encoding the mouse PTEN gene was synthesized from GENEWIZ (Suzhou, China). pAAV-CAG-PTEN was obtained by replacing mCherry gene in pAAV-CAG-mCherry with PTEN gene. Reverse the PTEN gene sequence in pAAV-CAG-PTEN to get pAAV-CAG-PTEN^R.
The anti-PD-1 used in this study is AUNP-12, a polypeptide purchased from Selleck (USA) with a purity of 99.20%. Cy5-AUNP-12 was purchased from Guoping pharmaceutical co (Anhui, China) with a purity of 90%. CpG was synthesized in GENEWIZ (Suzhou, China) with 90% purity.
Antibodies for Western blot (WB) and Immunofluorescence (IF): anti-mouse PTEN, CRT, β-actin and anti-rabbit FITC, CY3 fluorescent secondary antibodies were purchased from ABclonal Tech (Wuhan, China). Antibodies for flow cytometry test: CD11c-PE, CD80-FITC, CD86-APC, CD3-APC, CD8-FITC, CD4-APC-eflour780, CD69-PE, CD274-PE, CD11b-FITC, CD206-PE-Cy7, CD86-APC, IL-4-PE, IFN-γ-APC, TNF-α-eflour450, IL-2-PE5.5, CD4-APC-eflour780, CD44-APC, CD62L-PE were all purchased from Invitrogen (USA).

Zhang Y., Yang L., Ou Y., Hu R., Du G., Luo S., Wu F., Wang H., Xie Z., Zhang Y., He C., Ma C., Gong T., Zhang L., Zhang Z, & Sun X. (2023). Combination of AAV-delivered tumor suppressor PTEN with anti-PD-1 loaded depot gel for enhanced antitumor immunity. Acta Pharmaceutica Sinica. B, 14(1), 350-364.

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Comprehensive Immune Cell Profiling

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For Th1/Th2/Th17/Treg cell staining, spleen mononuclear cells were obtained and incubated with cell activation cocktail (00-4975-03, Invitrogen)at 37 °C for 5 h. Then the cells were incubated with Fc-block at room temperature for 10 min to avoid non-specific binding. To detect extracellular proteins, anti-mouse CD4-FITC (11-0041-82, 0.25 μg/test, Invitrogen) and anti-mouse CD25-APC (17-0251-81, 0.125 μg/test, Invitrogen). The True-Nuclear Transcription Factor Buffer Set (00-5523-00, Invitrogen) was applied before anti-mouse Foxp3-PE (12-5773-82, 1 μg/test, Invitrogen). For Th1/Th2/Th17 cell assay, PBMCs were stained with intracellular IFN-γ-APC (17-0251-81, 0.125 μg/test, Invitrogen), IL-4-PE (12-7041-82, 0.125 μg/test, Invitrogen), and IL-17- PerCP (45-7177-82, 0.03 μg/test, Invitrogen).The lymphocyte cells were tested with FACScan Flow Cytometry and WinMDI12.9 and analyzed by FlowJo X software. Each sample was set to analyze 50,000 cells. The gating strategy is provided in the Supplementary Fig. S8.

Gong B., Meng F., Wang X., Han Y., Yang W., Wang C, & Shan Z. (2024). Effects of iodine intake on gut microbiota and gut metabolites in Hashimoto thyroiditis-diseased humans and mice. Communications Biology, 7, 136.

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Multiparameter Flow Cytometry of Immune Cells

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The following monoclonal antibodies reactive with human species were used for staining: CD8-VioGreen (BW135/80, cat.: 130-113-726, 1:50), CD4-APC-Vio770 (VIT4, cat.: 130-113-211, 1:50), CD154/CD40L-PE-Vio770 (5C8, cat.: 130-096-793, 1:10), CD20-VioGreen (LT20, cat.: 130-113-379, 1:50), CD14-VioGreen (TÜK4, 130-113-153, 1:50) (all from Miltenyi Biotec, Bergisch Gladbach, Germany);
IL-13-FITC (PVM13-1, cat.: 11-7139-42, 1:20 or 85BRD, cat.: 11-7136-42, 1:20), IL-4-PE (8D4-8, cat.: 12-7049-42, 1:20) (all from Thermo Fisher Scientific); TNFa-Pacific Blue (MAb11, cat.: 502920, 1:100), IFNg-PerCp-Cy5.5 (4S.B3, cat.: 502526, 1:20), CD3-APC (Okt3, cat.: 17-0037-42, 1:50) (all from Biolegend, San Diego, CA, US). Fixation and permeabilization were performed using the Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s suggestions followed by intracellular cytokine and CD154/CD40-L staining. Fixable viability dye was used in eFluor-506 (cat.: 65-0866-14, 1:1000, Thermo Fisher Scientific).
Cells were acquired using BD FACSCanto II (with Diva software, Heidelberg, Germany) and post-acquisition data analysis was carried out using FlowJo software (TreeStar, Ashland, OR, US).

Ebner F., Morrison E., Bertazzon M., Midha A., Hartmann S., Freund C, & Álvaro-Benito M. (2020). CD4+ Th immunogenicity of the Ascaris spp. secreted products. NPJ Vaccines, 5, 25.

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FACS Analysis of Immune Cell Subsets

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Single cells isolated from spleen and intestinal lymph node were stained for FACS analysis as described previously (16 (link)). Lymphocytes were stained using the Zombie Violet™ Fixable Viability Kit (BioLegend Biotechnology Co., LTD, China) and were stained for surface markers including CD3-APC-Cy7 and CD8-APC (Thermo Fisher Scientific Co., LTD., USA); cells were then fixed and permeabilized by the Intracellular Fixation and Permeabilization Buffer Set (Thermo Fisher Scientific Co., LTD., USA) and stained for intracellular expression markers such as IFN-γ-PerCP and IL-4-PE (Thermo Fisher Scientific Co., LTD., USA). DCs were stained for surface markers including CD11c+-PE-Cy7, CD80-APC, CD86-FITC, and MHC-II-PE (Thermo Fisher Scientific Co., LTD., USA). Data were acquired with BD FACSVerse (Becton Dickinson Co., LTD., Canada) and analyzed with BD FACSuite (Becton Dickinson Co., LTD., Canada).

Tian X., Fan R., He H., Cui Q., Liang X., Liu Q., Liu T., Lin K., Zhang Z., Yi H., Gong P, & Zhang L. (2022). Bifidobacterium animalis KV9 and Lactobacillus vaginalis FN3 alleviated β-lactoglobulin-induced allergy by modulating dendritic cells in mice. Frontiers in Immunology, 13, 992605.

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FACS Analysis of Myeloid-Derived Suppressor Cells

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Cells were stained with specific antibodies or control isotypes using conventional protocols. The following anti-mouse antibodies were used for FACS analysis of MDSCs: CD11b/PECy5, Gr1/PE, Gr1/APC, CD62L/FITC, F4/80/FITC, CD11c/PE, and MHC CI OVA peptide/Biotin from eBiosciences (San Diego, CA); as well as CD124/PE, CD40/FITC, CD86/FITC, I^A-I^E/FITC, Ly6C/FITC and Ly6G/PE that were purchased from BD Biosciences (San Jose, CA). Lymphocytes characterization was performed with the next specific antibodies: CD8/PE and CD8/Biotin, CD69/PECy5, CD62L/FITC, CD247/PE, CD4/PE, CD4/PECy5, CD25/PECy5, Foxp3/Biotin, IFN-γ/FITC, IL-4/PE and IL-17/PE, all from eBiosciences. FITC-conjugated Streptavidin was purchased also from eBiosciences. In all cases, cells were previously incubated with anti-mouse FcγR 2.4G2 ascites (HB-197; ATCC) to reduce the non-specific binding. For intracellular staining, cells were fixed and permeabilized, according to the manufacturer’s protocol, with either Foxp3 Staining Buffer Set or IC Fixation and 10X Permeabilization Buffers from eBiosciences. Cells were acquired using both FACScan (BD Biosciences) and Gallios (Beckman Coulter, Miami, FL) flow cytometers and analyzed with Kaluza 1.2 (Beckman Coulter) and FlowJo 5.7.2 (Tree Star Inc., USA) softwares.

Fernández A., Oliver L., Alvarez R., Hernández A., Raymond J., Fernández L.E, & Mesa C. (2014). Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells. Journal for Immunotherapy of Cancer, 2, 5.

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Flow Cytometry Immunophenotyping Protocol

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For flow cytometry, the following fluorochrome-conjugated monoclonal antibodies were used. BD Biosciences: HLA-DR-APC (Clone: G46-6), CD86-FITC (Clone: FUN-1), CD80-PE (Clone: L307.4), CD54-APC (Clone: HA58), CD25-FITC (Clone: M-A251), CD127-BV421 (clone HIL-7R-M21), IFN-γ-FITC (Clone: 4S.B3), IL-4-PE (Clone: MP4-25D2); eBioscience: FoxP3-APC (Clone: 236A/E7), IL-17A-PE (Clone: Ebio64cap17); Beckman Coulter: CD40-PE (Clone: MAB89); Biolegend: CD4-PerCP (Clone: SK3). Cell viability was detected using the fixable viability dye eFluor 506 (eBioscience).
Antigen affinity-purified polyclonal anti-human TLR4 goat IgG was purchased from R&D systems. Cytokines (recombinant human granulocyte-macrophage colony-stimulating factor and IL-4), MicroBeads (CD14 and CD4) and cell purification units were obtained from Miltenyi Biotec. Protein-A agarose beads were from Cell Signalling Technology, and E. coli 055:B5 LPS and Polymyxin B-conjugated agarose beads were from Sigma-Aldrich. TLR4 signaling inhibitor CLI-095 and CpG ODN 2006 were procured from InvivoGen.

Mukherjee S., Karnam A., Das M., Babu S.P, & Bayry J. (2019). Wuchereria bancrofti filaria activates human dendritic cells and polarizes T helper 1 and regulatory T cells via toll-like receptor 4. Communications Biology, 2, 169.

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Flow Cytometric Analysis of T Cell Subsets

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To determine the Th1, Th2, Th17, and Treg cells in mice, flow cytometric analysis was performed on isolated DRG cells using CD4, Foxp3, IL-17A, TGF-β, and IL-4 antibodies as reported previously (18 (link)). Briefly, the cells suspension was transferred into 1 mL phosphate buffer saline (PBS) and centrifuged at 350 g for 5 min. After centrifugation, the supernatant was removed and the cells were resuspended with 500 μL fixation/permeabilization then centrifuged at 350 g for 5 mice after standing at room temperature for 30 min. The resuspension was repeated for twice. The cells were then incubated with monoclonal antibodies, including CD4-FITC, FOXP3-PE, IL-17A-PE, IL-4-PE, and IFN-γ-PE antibodies (eBiosciences, San Diego, California, USA) at dark for 30 min. After washing with PBS, the cells were resuspended in 150 μL PBS and then tested by using Beckman counter flow cytometer (USA). The data were analyzed using FlowjoX software. The lymphocytes were gated by FSC and SSC. CD4⁺IL-17A⁺, CD4⁺IL-4⁺, CD4⁺ IFN-γ⁺, and CD4⁺ FOXP3⁺ lymphocytes were identified as Th17, Th2, Th1, and Treg respectively.

Wang Z., Wu H., Chen Y., Chen H., Wang X, & Yuan W. (2021). Lactobacillus paracasei S16 Alleviates Lumbar Disc Herniation by Modulating Inflammation Response and Gut Microbiota. Frontiers in Nutrition, 8, 701644.

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Multiparametric Immune Cell Analysis

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All antibodies were purchased from commercial sources: CD3-phycoerythrin (PE), CD4-allophycocyanin (APC), CD4-peridinin-chlorophyll–protein complex (PerCP), CD19-PE, CD3-APC, CD3-fluorescein isothiocyanate (FITC), CD8-PE, NK1.1-APC, F4/80-PE, CD11c-PE, CD11c-APC, IL-4-PE, IFN-γ-APC, TNFα-FITC, CD69-APC, anti-CD3 Abs, and anti-CD28 Abs from eBioscience; Rabbit polyclonal GIT2 Abs, anti-phospho-PAK1/2 (Ser199/204 of PAK1 and Ser192/197 of PAK2 Abs, and anti-phospho-c-Cbl (Y774) Abs from Cell Signaling; Anti-phospho-Erk1/2 (E4) Abs, anti-PAKα, anti-Erk1/2 (K23) from Santa Cruz.

Hao Y.E., He D.F., Yin R.H., Chen H., Wang J., Wang S.X., Zhan Y.Q., Ge C.H., Li C.Y., Yu M, & Yang X.M. (2015). GIT2 deficiency attenuates concanavalin A-induced hepatitis in mice. FEBS Open Bio, 5, 688-704.

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Comprehensive Immunological Signaling Analysis

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POSH, JIP-1, JNK1, JNK2, Rac1, Noxa, MKK7, MLK3, MEKK1, and p-NFATc1 were purchased from Santa Cruz Biotechnology. Tak1, Puma, pSAPK/JNK, pMKK7, p-p38, pIκBα, NFATc1, JunB, Bcl2 and Bim were purchased from Cell Signaling. CD25 APC, T-bet APC, GATA3 PE, IL-4 PE, IFN-γ APC, IL-2 APC, and IL-17A APC were purchased from eBioscience. β-actin was purchased from Sigma. 4G10, Rac1 and Rac2 were purchased from Millipore. Mcl-1 was purchased from Rockland. IL-12Rβ2 PE was purchased from R&D. 7-AAD was purchased from BD.

Cunningham C.A., Cardwell L.N., Guan Y., Teixeiro E, & Daniels M.A. (2016). POSH Regulates CD4+ T cell Differentiation and Survival. Journal of immunology (Baltimore, Md. : 1950), 196(10), 4003-4013.

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Comprehensive Immunophenotyping of T and B Cells

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Phenotypic analyses of T cells and B cells were performed with anti-human monoclonal antibodies (mAbs): anti-human CD3-PerCP, CD4-FITC, CD19-PerCP and CD21-APC were from BD Biosciences (San Jose, CA, USA). CXCR5-APC, ICOS-PE, PD-1-PE, IFN-γ-PE, IL-4-PE, IL-17-PE, IL-21-PE, IL-22-PE, CD27-FITC, CD86-PE, CD95-PE, CD25-APC, and Foxp3-PE antibodies were obtained from eBiosciences (San Diego, CA, USA). Cells were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA) and data was analyzed by FlowJo software (Tree Star, San Carlos, CA).

Kong F., Zhang W., Feng B., Zhang H., Rao H., Wang J., Cong X, & Wei L. (2015). Abnormal CD4 + T helper (Th) 1 cells and activated memory B cells are associated with type III asymptomatic mixed cryoglobulinemia in HCV infection. Virology Journal, 12, 100.

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