Il 4 bv605

Manufactured by BioLegend

IL-4-BV605 is a fluorescently labeled antibody produced by BioLegend. It binds to the interleukin-4 (IL-4) cytokine, which plays a role in immune regulation. The BV605 fluorescent dye is used to label the antibody, allowing for detection and analysis in flow cytometry applications.

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4 protocols using il 4 bv605

Intracellular Staining and Cytokine Detection

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For intracellular staining of phosphor-proteins, cells were fixed with 2% paraformaldehyde, followed by permeabilization in 95% methanol. The following antibodies were used: anti-p-PDHE1α (AP1062; Calbiochem, Darmstadt, Germany) and a fluorescein isothiocyanate conjugated antirabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX). For staining of intracellular cytokines/transcription factors, either the Mouse Foxp3 Buffer Set 560409 (BD Biosciences, Franklin Lakes, NJ) or the Foxp3/Transcription Factor Staining Buffer Set 00-5523-00 (eBioscience, San Diego, CA) was used along with the following antibodies: CD45-BV421, CD3-PECy7, CD4-APC, CD8-Alexafluor700, NK1.1-BV711, IFN-γ-BV510, IL-4-BV605, IL-17-PerCP-Cy5.5, CD25-BV785, Foxp3-FITC, CD69-PerCPCy5.5, and CD62L-PerCPCy5.5 (all from BioLegend, San Diego, CA). Cell viability was measured using a Live/Dead staining kit (Thermo Fisher). Cell death was measured using an FITC/Annexin V Apoptosis Detection Kit (556547; BD Biosciences).

Lee H., Jeon J.H., Lee Y.J., Kim M.J., Kwon W.H., Chanda D., Thoudam T., Pagire H.S., Pagire S.H., Ahn J.H., Harris R.A., Kim E.S, & Lee I.K. (2022). Inhibition of Pyruvate Dehydrogenase Kinase 4 in CD4+ T Cells Ameliorates Intestinal Inflammation. Cellular and Molecular Gastroenterology and Hepatology, 15(2), 439-461.

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Multiparameter Flow Cytometry of Immune Cells

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Cells in BALF were stained with CD45-FITC (clone 30-F11), CD11b-APC (clone M1/70), CD11c-PE (clone N418), F4/80-PE-CY7 (clone BM8), and Ly6G-BV421 (clone 1A8, BioLegend) in the presence of an Fc blocker (CD16/CD32, BD Biosciences). A total of 2 × 10⁶ single lung cells were stimulated with cell stimulation cocktail and protein transport inhibitor cocktail (Cat#00-4970-03 and #00-4980-93, eBioscience) in IMDM medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin for 4 h in a 37°C incubator with 5% CO₂. The cells were incubated with a Zombie NIR™ Fixable Viability Kit (Cat#423105, BioLegend) at room temperature for 15 min and then stained with antibodies to extracellular antigens in the presence of Fc blocker at 4°C for 25 min. Intracellular and nuclear staining was performed with the Foxp3/Transcription buffer set (Cat# 00-5523-00, eBiosciences). Data were acquired using LSRFortessa and FACSDiva software (BD Biosciences) or Attune NxT 3 L-BRV and Attune NxT software (Thermo Fisher Scientific) and analyzed using FlowJo 10.7.2 (TreeStar, Ashland). The following antibodies were used: CD4-FITC (clone GK1.5), TCR-BV421 (clone H57-597), Foxp3-AF647 (clone MF23), IL-10-PE (JES5-16E3, BD Biosciences), IFN-γ-PE-Cy7 (clone XMG1.2), IL-4-BV605 (clone 11B11), IL-17a-BV510 (clone TC11-18H10.1, Biolegend), and IL-13-PE-eFluor 610 (eBiosciences).

Li J.D, & Yin J. (2023). Interleukin-10-alveolar macrophage cell membrane-coated nanoparticles alleviate airway inflammation and regulate Th17/regulatory T cell balance in a mouse model. Frontiers in Immunology, 14, 1186393.

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Characterization of Immune Cell Activation

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In order to examine activation status of the cells, BMDCs were treated with Duolac ATP or LPS for 24 h at 37°C. The cells were stained with anti-mouse CD86-FITC, PD-L1-PE, MHC II-PE-cy7, CD11c-APC (all from BD Biosciences) for 20 min at 4°C in the dark. To test the increase of Treg, CTV labeled CD4⁺ T cells cultured with Duolac-treated BMDCs for 3 days were stained with anti-mouse CD4-PE (BD Biosciences). After surface staining, CD4⁺ T cells were fixed and stained with anti-mouse Foxp3-APC mAb (BioLegend, Dedham, MA, United States) using FOXP3 Fix/Perm Buffer Set (BioLegend). In vivo examination, single cells from mLNs and PP were isolated from AD mice. Population changes of DCs and Tregs were examined as aforementioned. To analyze for subpopulation of Th cells, total mLN and PP cells were stimulated with PMA and ionomycin (Sigma-Aldrich) in the presence of brefeldin A for 4 h. After the stimulation, the cells were stained with appropriate combination of anti-mouse CD11c-APC, CD4-bv605, Foxp3-APC, IFN-gamma-PE, IL-4-bv605, and IL-17-APC-cy7 mAb (all from Biolegend). The cells were washed and the expression was examined using a FACSCanto II (BD Biosciences). All flow cytometric data acquired were analyzed with FlowJo software (Tree Star, Ashland, OR, United States).

Kim H.W., Hong R., Choi E.Y., Yu K., Kim N., Hyeon J.Y., Cho K.K., Choi I.S, & Yun C.H. (2018). A Probiotic Mixture Regulates T Cell Balance and Reduces Atopic Dermatitis Symptoms in Mice. Frontiers in Microbiology, 9, 2414.

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Tfh and Tfreg Cell Analysis in PBMCs

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A total of 1.5x10 6 and 2x10 6 fresh PBMCs were used for Tfh and Tfreg analysis, respectively. PBMCs were stained with a fixable viability dye-700-alexa fluorophore at 4 0 C for 15 minutes in the dark (for dead cell discrimination) prior to surface and intracellular staining. PBMCs were subsequently stained with surface markers at 4 0 C for 30 minutes in the dark with the following monoclonal antibodies: anti-CD3-PE-cy7, anti-CD4-Buv496, anti-CD8-FITC, anti-CXCR5-Percp-cy5.5, anti-PD-1-BV786, anti-CXCR3-PE, anti-CCR6-BV711, and anti-CD25-APC-cy-7. All monoclonal antibodies were purchased from BD Bioscience. For intracellular cytokine staining (ICS), PBMCs were stimulated for 5 hours with phorbol 12-myristate 13acetate (PMA) (50 ng/mL), ionomycin (500 ng/mL) (Sigma-Aldrich), and golgistop (monensin; 0.6 μL/mL) (BD Bioscience). Following stimulation, PBMCs were fixed and permeabilized with cytofix/cytoperm cell permeabilization buffer (BD Bioscience). ICS was then performed using monoclonal antibodies against IFNγ-APC, IL-17-BV650, IL21-BV421, IL-10-PE-CF594 (BD Bioscience) and IL-4-BV605 (Bio-legend) following incubation under the same conditions described above for surface staining. Simultaneously, FoxP3 staining was performed for 30 minutes in dark at 4 0 C using anti-FoxP3-PE antibody according to the manufacturer's instructions (eBioscience).

Haque R., Kim Y., Jang H., Kim S.Y., Lee H., & Kim H.J. (2020). Dysregulated proinflammatory circulating follicular helper T cells and suppressive follicular regulatory T cells in patients with multiple sclerosis.

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