Anti cd4 efluor 450

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-CD4 eFluor 450 is a fluorescently-labeled antibody used for the detection and quantification of CD4-expressing cells in flow cytometry applications. It is designed to bind to the CD4 surface marker, which is expressed on a subset of T lymphocytes.

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5 protocols using anti cd4 efluor 450

Flow Cytometric Identification of Th17 Cells

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Murine splenocytes were incubated with allophycocyanin (APC)-conjugated anti-CD4 antibodies (BD Biosciences) and then permeabilized using a Foxp3 Staining Buffer Set (eBioscience, San Diego, CA, USA). To identify Th17 cells, murine CD4⁺ T cells were stained with anti-RORγt antibodies conjugated with phycoerythrin or anti-IL-17 antibodies conjugated with fluorescein isothiocyanate (both from eBioscience). For detection of IL-17, cells were incubated with 5 ng/mL of phorbol- 12-myristate-13-acetate (PMA), 500 ng/mL ionomycin, and 1 µL/mL of GolgiPlug (BD Biosciences) for 4 h prior to staining with anti-IL-17 antibodies.
Human Th17 cells were identified based on co-expression of RORγt, and IL-17. PBMCs were incubated with 50 ng/mL of PMA, 250 ng/mL of ionomycin, and 1 µL/mL of GolgiPlug for 4 h. After incubation with anti-CD4 eFluor 450 (eBioscience), cells were permeabilized using a Foxp3 Transcription Factor Staining Buffer Set (eBioscience). RORγt and IL-17 expression was detected by staining with anti-RORγt antibodies conjugated with phycoerythrin and anti-IL-17A antibodies conjugated with APC (both from eBioscience). After staining with antibodies, the cells were assessed on an LSRFortessa cell analyzer (BD Biosciences). The acquired data were analyzed using FlowJo 7.6.5 software (TreeStar Inc., Ashland, OR, USA).

Jung S.M., Kim Y., Kim J., Jung H., Yi H., Rim Y.A., Park N., Kwok S.K., Park S.H, & Ju J.H. (2018). Sodium Chloride Aggravates Arthritis via Th17 Polarization. Yonsei Medical Journal, 60(1), 88-97.

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Comprehensive Murine Immune Cell Profiling

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Mouse cells were preincubated with purified antimouse CD16/CD32, and human cells were incubated with FcR-blocking reagent (BD Biosciences). Isolated cells were stained with labeled antibodies in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA). Dead cells were excluded based on staining with Live/Dead fixable dye (eBioscience). Forkhead box P3 (FOXP3) fixative solution (eBioscience) was used for FOXP3 staining. Prepared samples were analyzed using a flow cytometer (FACSCalibur or FACSAria; BD Biosciences). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The following beads and antibodies were used: OneComp eBeads (eBioscience), antimouse CD11c Brilliant Violet 421 (BioLegend), antimouse CD11b PE-cy7 (eBioscience), GR1-APC (eBioscience), LY6C-APC-eFluor (eBioscience), F4/80 PE (eBioscience), NK-1.1 Percp-Cy5.5 (eBioscience), anti-CD45 FITC (eBioscience), Live/Dead Aqua (eBioscience), anti-CD4 eFluor 450 (eBioscience), anti-CD8 APC eFluor 780 (eBioscience), anti-CD3 Percp-Cy5.5 (eBioscience), anti-CD19 PE-CY7 (eBioscience), anti-CD25 APC (eBioscience), anti-Foxp3 PE (eBioscience), and anti-CD3 APC (BioLegend).

Zhang H., Jiang R., Zhou J., Wang J., Xu Y., Zhang H., Gu Y., Fu F., Shen Y., Zhang G., Feng L., Zhang X., Chen Y, & Shen F. (2020). CTL Attenuation Regulated by PS1 in Cancer-Associated Fibroblast. Frontiers in Immunology, 11, 999.

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Multiparametric Flow Cytometry Analysis of Primary T Cell Subsets

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The phenotype of freshly-isolated primary T cell subsets was evaluated by multiparameter flow cytometry. Cells were incubated in the dark for 20 min at room temperature (RT) with a panel of labeled monoclonal antibodies (mAbs): anti-CD4-eFluor 450 (48-0048-42, eBioscience, CA, USA), CD8-ECD (6604728, Beckman Coulter, Brea, CA, USA), CD39- PC7(25-0399-42, eBioscience) PD-1-PE (12-2799-42, BioLegend), CD25-APC/Cy7 (302613, BioLegend, San Diego, CA, USA); CD69 FITC (555530, BD Biosciences, San Jose, CA, USA) and Foxp3-APC (17-4776-41, eBioscience). Isotype controls were used for each experiment. After incubation, cells were again washed, resuspended in flow buffer and analyzed using Gallios flow cytometer (Beckman Coulter). At least 5 × 10⁴ events were collected, and the data were analyzed using Kaluza software (Beckman Coulter). For fluorescence quantitation standardized fluorochrome microspheres were used as recommended by the company (Quantum^TM FITC-5 MESF, 555, Bangs Laboratories, Fishers, Indiana, USA).

Muller L., Mitsuhashi M., Simms P., Gooding W.E, & Whiteside T.L. (2016). Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets. Scientific Reports, 6, 20254.

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Multiparametric Flow Cytometry Analysis

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Flow cytometry was performed on a Miltenyi Macsquant Analyzer. Antibodies used included anti-CD4 eFluor450 (eBioscience 48-0041-82), anti-CD25 PE (BD Biosciences 553866), anti-CD62L PE (eBioscience 12-0621-82), anti-CD69 PE (BD Biosciences 553237), anti-EZH2 PE (BD 562478), anti-FoxP3-APC (eBioscience 17-5773-80), anti-FoxP3-PE (eBioscience 12-5773-80), anti-Helios PE (eBioscience 12-9883-41), anti-ICOS PE (eBioscience 12-9942-81), anti-ID3 PE (BD Biosciences 564564), anti-mouse IFN gamma APC (eBioscience 17-7311-81), anti-mouse IL2 PE (BD Bioscience 561061), anti-KI67 eFluor450 (eBioscience 48-5698-80). Flow cytometry antibodies were used at a 1:100 dilution except for anti-EZH2 PE, anti-FoxP3-APC, anti-FoxP3-PE, anti-Helios PE, anti-ICOS PE, anti-ID3 PE, anti-mouse IFN gamma APC, anti-mouse IL2 PE, anti-KI67 eFluor450, which were all used at a 1:50 dilution. Cell trace violet (Invitrogen #C34557) was used following manufacturer’s specifications. Flow cytometry data analysis was performed using FlowJo.

Gerriets V.A., Kishton R.J., Johnson M.O., Cohen S., Siska P.J., Nichols A.G., Warmoes M.O., de Cubas A.A., MacIver N.J., Locasale J.W., Turka L.A., Wells A.D, & Rathmell J.C. (2016). Foxp3 and Toll-like receptor signaling balance Treg cell anabolic metabolism for suppression. Nature immunology, 17(12), 1459-1466.

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Intracellular cAMP Levels in Tregs

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PBMCs from 10 PR3-positive GPA patients and 10 matched HCs (cAMP cohort, Table 1) were stimulated with anti-CD3/anti-CD28 Dynabeads. Intracellular cAMP levels in _MTregs were assessed by flow cytometry. First, cells were fixed and permeabilized using a FoxP3 Fixation and Permeabilization kit (eBioscience) according to the manufacturer's protocol. Next, cells were stained with an unconjugated mouse-anti-cAMP antibody followed by a secondary goat-anti-mouse-PE antibody (Abcam, Cambridge, UK). _MTregs were stained using anti-CD3-PerCP, anti-CD4-eFluor450, anti-CD45RO-FITC, and anti-FoxP3-APC (eBioscience) and analyzed on a BD LSRII flow cytometer (Becton-Dickinson). Data were analyzed using Kaluza software (V1.5a, Beckman Coulter), and _MTregs were defined as CD3⁺CD4⁺CD45RO⁺FoxP3^high.

Dekkema G.J., Bijma T., Jellema P.G., Van Den Berg A., Kroesen B.J., Stegeman C.A., Heeringa P., Abdulahad W.H, & Sanders J.S. (2019). Increased miR-142-3p Expression Might Explain Reduced Regulatory T Cell Function in Granulomatosis With Polyangiitis. Frontiers in Immunology, 10, 2170.

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