Anti igd fitc

Manufactured by BioLegend

Sourced in United Kingdom

Anti-IgD-FITC is a fluorescently-labeled antibody that binds to the IgD receptor. It is used in flow cytometry and other immunoassays to detect and quantify IgD-expressing cells.

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4 protocols using anti igd fitc

Comprehensive flow cytometry protocol

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FACS staining was external unless otherwise specified. Cells were stained for LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm Excitation (Invitrogen, UK). Internal staining: cells were fixed with 2% formaldehyde (Sigma Aldrich, UK) and permeabilized with IX permeabilization buffer (eBioscience, UK). The following antibodies were used for flow cytometric analysis: anti-CD4-PerCp-Cy5.5 (RPA-T4), anti-CD19-PB (HIB19), anti-CD38-PerCP-Cy5.5 (HIT2), anti-IgD-FITC (IA6-2), anti-CXCR5-PB (J252D4), anti-CD27-PE-Cy7 (M-T271), anti-CD34-FITC (561), anti-CD68-APC (Y1/82A), anti-CD83-BV421 (HB15e), anti-ICOS-APC (C398.4A) (Biolegend, UK), anti-CD20-PO (HI47, Life technologies, UK), anti-CD138-APC (B-A38, Beckman Coulter, UK), anti-CD10-PE-Cy7 (HI10a), anti-CD45-RA-FITC (T6D11), anti-CD161-PE (191B8), anti-IgM-APC (PJ2-22H3), anti-CXCR4-APC (12G5, Milteny Biotec, UK), anti-CD3-FITC (ICHT1, R&D Systems, UK), anti-LLT1-PE (402659 R&D), anti-IgG isotype control-PE, IgG1 and IG2A isotype controls (R&D Systems, UK), anti-mouse IgG-PE (R&D Systems, UK).
Novel mouse anti-human CLEC2D (LLT1) monoclonal antibodies were generated, clones 359.7G7, mIgG1 and 359.2H7, mIgG2al. All purified antibodies are dialyzed against PBS, are low in endotoxin (< 2EU/mg), and are filtered sterile.

Llibre A., López-Macías C., Marafioti T., Mehta H., Partridge A., Kanzig C., Rivellese F., Galson J.D., Walker L.J., Milne P., Phillips R.E., Kelly D.F., Freeman G.J., El Shikh M.E., Klenerman P, & Willberg C.B. (2016). LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation. The Journal of Immunology Author Choice, 196(5), 2085-2094.

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Isolation and Characterization of SARS-CoV-2-Specific Memory B Cells

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B cells were isolated from PBMCs of convalescent individuals with COVID-19 using immunomagnetic negative selection (EasySep Human B Cell Enrichment Kit, STEMCELL, 17954). In this process, Tetrameric Antibody Complexes and dextran-coated magnetic particles targeted and removed non-B cells. Following isolation, B cells underwent staining with anti-CD19-APC (BioLegend, 302212), anti-IgD-FITC (BioLegend, 348206), anti-IgM–FITC (BioLegend, 314506) phenotyping antibodies, and biotinylated SARS-CoV-2 (BA.4/5) Spike RBD antigen. Viability was assessed using 7-AAD Stain (Invitrogen, 00699342). Class-switched memory B cell-antigen complexes (CD19⁺Ag⁺IgM^-IgD^-7-AAD^-) were then detected with a PE-labeled streptavidin conjugate (BioLegend,127807), and target memory B cells were isolated via flow-cytometric sorting using a BD FACSAira Fusion (BD Biosciences). Subsequently, cells were cultured and activated in human B cell expansion medium for 8 days (ImmunoCultTM Human B Cell Expansion Kit, STEMCELL, 1000645). Flow-cytometric data analysis was conducted using FlowJo version 10.8.1 (Tree Star).

Yu P., Ran J., Yang R., Zhu H., Lu S., Wu Y., Zhao T, & Xiong T. (2024). Rapid isolation of pan-neutralizing antibodies against Omicron variants from convalescent individuals infected with SARS-CoV-2. Frontiers in Immunology, 15, 1374913.

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Quantifying SARS-CoV-2-specific Memory B Cells

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SARS-CoV-2-specific memory B cells were detected as we described previously^{11 (link)}. First, biotinylated antigens were multimerized with fluorescence-labeled streptavidin individually. Recombinant spike protein (R&D, #BT10549-050) was mixed with BV510-streptavidin (BioLegend) at 10:1 ratio and BV785-streptavidin (BioLegend) at 18:1 ratio at 4 °C for 1 h. Recombinant RBD protein (R&D, #BT10500-050) was mixed with BV421-streptavidin (BioLegend) at 20:1 ratio at 4 °C for 1 h. The antigen probes prepared above were then mixed in 50 mM free d-biotin (Macklin) in PBS to ensure minimal cross-reactivity. PBMCs were thawed and let to rest at 37 °C with 5% CO₂ for 2 h and stained with Zombie Red (BioLegend) in PBS at room temperature for 20 min. Cells were then stained with 50 μL of antigen probe cocktail containing 100 ng of spike and 50 ng of RBD at 4 °C for 30 min. Cells were washed with PBS and then stained with the following antibody cocktail: anti-CD3-Pacific Blue™, anti-CD19-PE-CY7, anti-CD27-AF700, anti-IgD-FITC, anti-CD38-BV650, anti-IgM-BV605, anti-IgG-AF647, anti-IgA-PE all from BioLegend at 1:100 dilution. Samples were acquired by flow cytometry.

Liu Y., Zeng Q., Deng C., Li M., Li L., Liu D., Liu M., Ruan X., Mei J., Mo R., Zhou Q., Liu M., Peng S., Wang J., Zhang H, & Xiao H. (2022). Robust induction of B cell and T cell responses by a third dose of inactivated SARS-CoV-2 vaccine. Cell Discovery, 8, 10.

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Tonsil B Cell Immunophenotyping

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Tonsil mononuclear cells were thawed, washed, and counted as described above. To stain apoptotic and necrotic cells, cells were incubated with Zombie NIR fixable viability dye (Biolegend) for 15 min at room temperature. Cells were washed, resuspended in a surface stain cocktail containing anti-IgD-FITC, anti-CD38-PE, anti-CD27-PE/Cy7, anti-CD19-APC, anti-CD3-APC/Cy7, and anti-CD14-APC/Cy7 (all from Biolegend), and incubated for 45 min on ice. Subsequently, cells were washed two times, passed through a 35 µm nylon mesh and sorted on a BD FACS Aria II at the Harvard Microbiology and Immunobiology (MBIB) Flow Cytometry Core. Lymphocytes were electronically gated according to their forward scatter and side scatter properties, negatively gated to exclude viability dye⁺, CD3⁺, and CD14⁺ cells, and positively gated for B cells (CD19⁺). B cell subsets were subsequently gated as follows: IgD ⁺CD27^- cells (Naive); IgD^- CD38^hi CD27^+/- (GC); IgD^- CD38^lo/mid CD27 ⁺ (Memory) (See also Fig. 1b and Supplementary Fig. 1). The CD27 gate was set based on a PE/Cy7 FMO gating control. Sorted cells were pelleted, washed 2× with PBS, then lysed in Buffer RLT (Qiagen, for RNA analysis), 2% NP-40 lysis buffer (for western blot), or snap frozen in an isopropanol/dry ice slurry (for glycomic analysis).

Giovannone N., Liang J., Antonopoulos A., Geddes Sweeney J., King S.L., Pochebit S.M., Bhattacharyya N., Lee G.S., Dell A., Widlund H.R., Haslam S.M, & Dimitroff C.J. (2018). Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans. Nature Communications, 9, 3287.

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