CD4+ T cells were isolated and purified from the spleen and draining lymph nodes of EAU mice by positive selection using PE-conjugated anti-mouse CD4 antibody (BioLegend, San Diego, CA, USA), anti-PE microbeads, and auto-MACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. CD4+ T cells were maintained in RPMI 1640 medium (Gibco, CA, USA) with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA), 50 μM β-mercaptoethanol (Gibco), 2mM L-Glutamine (Gibco), and 100 U/mL Penicillin-Streptomycin (Gibco), referred to as complete RPMI 1640 medium. Then, CD4+ T cells (1 × 106 cells/well) were co-cultured with 1 × 106 irradiated (30 Gy) syngeneic splenocytes as APCs, which were pre-incubated with 10 μg/ml IRBP1-20 for 20 min in a 24-well plate, under Th17 cell polarization (culture medium supplemented with 10 ng/ml IL-23) or Th1 cell polarization (culture medium supplemented with 10 ng/ml IL-12).
Pe conjugated anti mouse cd4 antibody
Manufactured by BioLegend
Sourced in United States
The PE-conjugated anti-mouse CD4 antibody is a fluorescently labeled monoclonal antibody that specifically binds to the CD4 protein expressed on the surface of mouse T helper cells. This antibody can be used in flow cytometry applications to identify and quantify CD4-positive cell populations.
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Lab products found in correlation
Automacs separator, by Miltenyi Biotec (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Collagenase, by Merck Group (1 mentions)
Apc conjugated anti mouse cd3 antibody, by BioLegend (1 mentions)
Percp cyanine5.5 conjugated anti mouse cd8a antibody, by BioLegend (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
4 protocols using pe conjugated anti mouse cd4 antibody
1
Isolation and Polarization of CD4+ T Cells
2
Tumor-Infiltrating T-Cell Analysis
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Tumor-infiltrating T cells were analyzed by flow cytometry. First, tumor tissues were excised on day 27 after tumor re-challenge and digested in collagenase (1 mg/mL in RPMI; Sigma-Aldrich) for 2 h at 37 °C with gentle stirring. Red blood cells were lysed and the resulting tumor cell suspension was passed through a 40-μm strainer. The cells were then stained with the following fluorescent antibodies: APC-conjugated anti-mouse CD3 antibody (BioLegend), PE-conjugated anti-mouse CD4 antibody (BioLegend), and PerCP/cyanine5.5-conjugated anti-mouse CD8a antibody (BioLegend). The percentage of CD3(+)CD4(+) and CD3(+)CD8(+) cells was analyzed using a BD FACSCalibur flow cytometer, and CellQuest Pro and FlowJo software v10.0.7 (BD Bioscience).
3
Western Blotting and Flow Cytometry Analysis of CLEC18
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For Western blotting analysis, cells (1 × 106) were lysed by RIPA buffer, followed by fractionation on 12% SDS-PAGE before blotting onto PVDF membrane. Membranes were probed with anti-hCLEC18 mAb (clone 3A9E6 (1)) or anti-mCLEC18 mAb (home-made antibody C8G), followed by incubation with peroxidase-conjugated goat anti-mouse polyclonal antibody (Millipore, AP181P) or donkey anti-human polyclonal antibody (Jackson ImmunoResearch, 709-035-149). Blots were developed by the ImmobilonTM Western Chemiluminescent using horseradish peroxidase as the substrate (MilliporeTM). For flow cytometry analysis, primary cells were incubated with phycoerythrin (PE)-conjugated anti-mouse CD4 antibody (BioLegend, 100408), PerCP-Cy5.5-conjugated rat anti-mouse CD8a (BD, 561109), PE/cyanine7-conjugated anti-mouse CD11b antibody (BioLegend, 101216), Alexa Fluor 488-conjugated anti-mouse Ly6G/Ly6C (Gr-1) antibody (BioLegend, 108417), Brilliant Violet 510-conjugated anti-mouse IA/IE antibody (BioLegend, 107636), Brilliant Violet 421-conjugated anti-mouse/human CD45R/B220 antibody (BioLegend, 103251), and then intracellular stain with Alexa Fluor 647-conjugated anti-mCLEC18 mAb (home-made antibody clone C5F). After staining, cells were analyzed by a FACSVerse™ flow cytometer (BD Biosciences) and data were analyzed using the FlowJo software.
4
FoxP3 Expression in Nanoparticle-Treated T Cells
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The expression levels of FoxP3 in T cells after treatment with various nanoparticles were evaluated by flow cytometry. T cells were seeded onto 24-well plates (1 × 106 cells/well) and treated with various nanoparticles containing 20 μM fenofibrate for 48 h. Cells were harvested, washed with PBS and stained with FITC-conjugated anti-mouse CD3 antibody, PE/Cy5-conjugated anti-mouse CD25 antibody (Biolegend; catalog #102010, lot #B277468) and PE-conjugated anti-mouse CD4 antibody (Biolegend; catalog #116006, lot #B255181) for 1 h. Cells were fixed and permeabilized using a transcription factor buffer (BioLegend) and incubated with APC-conjugated anti-mouse FoxP3 antibody (Invitrogen; catalog #17-5773-82, lot #1984797) for 1 h.
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