Inguinal and popliteal lymph node were harvested and 12, 20, or 24 h post-immunization (p.i.), incubated 15 min with collagenase D (Roche) at 37°C and mechanically disrupted by glass-teasing. Cell suspensions were filtered through a 150 μm sieve and stain with biotinylated anti-mouse antibodies for lineage depletion (TCRβ, CD3ε, CD19, B220, NK1.1, Ly6G, PDCA-1) (Biolegend) for 20 min on ice. After washing, cells were incubated with anti-biotin microbeads (Miltenyi) for 15 min at 4°C. DC fraction enrichment was obtained by MACS negative selection (Miltenyi). After spin down, cells were stained with directly conjugated antibodies (CD45, CD11c, CD11b, I-Ab, lineage). Cells were acquired with LSR II Fortessa (BD) for analysis or facs-forted using FACSAria Fusion (BD). FlowJo (version 10, TreeStar) was used for post-acquisition analysis of the data.
Biotinylated anti mouse antibodies
Manufactured by BioLegend
Biotinylated anti-mouse antibodies are laboratory reagents used in various biomedical research applications. They are designed to specifically bind to mouse-derived protein targets, with the biotin label enabling further detection or interaction with streptavidin-based systems.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii fortessa, by BD (1 mentions)
Macs negative selection, by Miltenyi Biotec (1 mentions)
Anti biotin microbead, by Miltenyi Biotec (1 mentions)
Facsaria fusion, by BD (1 mentions)
Collagenase d, by Roche (1 mentions)
Pdca 1, by BioLegend (1 mentions)
Streptavidin peroxidase, by Merck Group (1 mentions)
2 protocols using biotinylated anti mouse antibodies
1
Isolation and Characterization of Lymph Node Dendritic Cells
2
Allergen-Specific Antibody Quantification
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MaxiSorp plates (Nunc A/S, Denmark) were coated with 0.5 µg purified Fes p 1 allergen in 50 μl 0.1 M sodium carbonate buffer (pH 9.6) overnight at 4°C. After blocking with PBS/2% BSA (1h, 37°C), the wells were incubated with 50 μl diluted mice sera (100 fold dilution for IgG, IgG1, IgG2, IgG3 and IgA and 6 fold dilution for IgE) for 1h at 37°C. Dilutions were made in PBS containing 1% BSA. Biotinylated anti-mouse antibodies (Biolegend, San Diego, CA) were used for specific antibody class/subclass determination. Streptavidin peroxidase (Sigma-Aldrich, St. Louis, Missouri, USA) was used for detection with ortho-phenylendiamin as the substrate.
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