Igd fitc

Manufactured by BioLegend

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IgD-FITC is a fluorescently labeled antibody that binds to the IgD receptor on the surface of B cells. It is used for the identification and analysis of IgD-expressing cells in flow cytometry applications.

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Anti-IgD-FITC (4 citations)

6 protocols using igd fitc

Single-cell Sorting of SARS-CoV-2 Spike-Reactive B Cells

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For single-cell sorting, PBMCs were treated with FcX blocking antibodies (BioLegend, #4422302) to reduce non-specific labeling of the cells. PBMCs were stained with S-trimer-Strep-tag, CD19-APC-Cy7 (BioLegend, #302217), and IgD-FITC (BioLegend, #348206) for 20 min on ice. After washing, cells were stained with Strep-Tactin XT-DY649 (IBA) for 20 min on ice. The cells were resuspended in FACS buffer (PBS containing 1% FCS, 1 mM EDTA, and 0.05% NaN3) supplemented with 0.2 μg/ml propidium iodide (PI) to exclude dead cells. Cell sorting was performed on Special Order System BD FACSAria II (BD Biosciences) to isolate S-trimer⁺ CD19⁺ IgD⁻ cells from the PI⁻ live cell gate. Cells were directly sorted into a 96-well PCR plate. Plates containing single-cells were stored at −80 °C until proceeding to RT-PCR. Flow cytometric data were acquired on BD LSRFortessa (BD Biosciences) or CytoFLEX S (Beckman Coulter). Flow cytometric data were analyzed using BD FACSDiva (v8.0.2, BD Biosciences), CytExpert software (v2.4, Beckman Coulter), or FlowJo software (v10.8.1, BD Biosciences).

Shitaoka K., Higashiura A., Kawano Y., Yamamoto A., Mizoguchi Y., Hashiguchi T., Nishimichi N., Huang S., Ito A., Ohki S., Kanda M., Taniguchi T., Yoshizato R., Azuma H., Kitajima Y., Yokosaki Y., Okada S., Sakaguchi T, & Yasuda T. (2023). Structural basis of spike RBM-specific human antibodies counteracting broad SARS-CoV-2 variants. Communications Biology, 6, 395.

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Multiparameter Analysis of Immune Cell Subsets

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Single-cell suspensions from spleens, bone marrow, or peripheral lymph nodes were blocked for 30 min using FBS. Cell-surface staining was achieved by incubating cells at 4°C for 30 min with fluorescence-conjugated anti–mouse antibodies (clone; source): XBP1s–Alexa Fluor 647 (Q3-695; BD), XBP1s-phycoerythrin (PE; Q3-695; BD), B220–Alexa Fluor 488 (RA3-6B2; BioLegend), B220-BV605 (RA3-6B2; BioLegend), CD43-PE (eBioR2/60; eBioscience), CD19–Alexa Fluor 647 (6D5; BioLegend), IgM-PE-Cy7 (RMM-1; BioLegend), IgD-FITC (11-26c.2a; BioLegend), GL7-PE (GL7; BioLegend), AA4.1-PE-Cy7 (AA4.1; BioLegend), CD1d-PerCP-Cy5.5 (1B1; BioLegend), CD23-FITC (B3B4; BioLegend), CD3-APC-Cy7 (145-2C11; BioLegend), CD4-BV605 (RM4-5; BioLegend), CD8α-PE-Cy7 (53–6.7; BioLegend), and CD138-PE (281–2; BioLegend). Viability staining was accomplished using DAPI exclusion during acquisition. Acquisition of B, T, and dendritic cell populations was performed on an LSRII cytometer (BD) harboring a custom configuration for the Wistar Institute. Cytometry data were analyzed using FlowJo software (7.6.1; Tree Star Inc.).

Tang C.H., Chang S., Paton A.W., Paton J.C., Gabrilovich D.I., Ploegh H.L., Del Valle J.R, & Hu C.C. (2018). Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization. The Journal of Cell Biology, 217(5), 1739-1755.

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Comprehensive Immune Cell Profiling

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Bone marrow, peripheral blood, and splenic B cells from cCD79 and wild type mice were analyzed using the following antibodies: hCD79A-PE (R&D Systems, FAB69201P); hCD79B-PE (abcam, ab33295); mCD79B-AF488 ([HM79], made in house); hCD79A-AF647 ([Curly-14], made in house); B220-BV786 (BD, 563894); B220-APC (BD, 103212); B220-PE (BD, 561878); CD43 ACP-Cy7 (BD, 562866); BP1-PE (BD, 553735); CD24-APC (BioLegend, 101814); IgD-FITC (BioLegend, 405704); IgM-BUV396 (BD, 564025); CD19-FITC (BioLegend, 152404); CD93-APC (BioLegend, 136510); CD23-FITC (BD, 561772); CD1d-PE (BD, 553846); CD95-PE (BD, 554258); GL7-FITC (BD, 553666); fab anti-mIgG (H+L)-AF647 (Jackson ImmunoResearch, 115–606-072); phospho-Syk (Y352)-PE (BD, 557881); PTEN-PE (BD, 560002); mouse IgG1 isotype control-PE (BD, 559320).

Wemlinger S.M., Parker Harp C.R., Yu B., Hardy I.R., Seefeldt M., Matsuda J., Mingueneau M., Spilker K.A., Cameron T.O., Larrick J.W., Getahun A, & Cambier J.C. (2022). Preclinical Analysis of Candidate Anti-Human CD79 Therapeutic Antibodies Using a Humanized CD79 Mouse Model. The Journal of Immunology Author Choice, 208(7), 1566-1584.

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SARS-CoV-2 Protein-Specific B-Cell Sorting

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For protein-specific B-cell sorting, cryopreserved B cells from five convalescent patients (rAbCOVID29, rAbCOVID27, rAbCOVID09, rAbCOVID07, and rAbCOVID19) were selected due to the availability of cells from these patients for this experiment (Figure S1). The cells were thawed, washed with Cell Staining Buffer (BioLegend, San Diego, CA, USA), and then incubated at 4°C for 20 min with a cocktail of antibodies and protein S1 and RBD (WT) tetramers. The cocktail consisted of CD19-BV421 (BioLegend clone HIB19), CD27-PeCy7 (BioLegend clone O323), IgD-FITC (BioLegend clone IA6-2), RBD-APC, and S1-PE tetramers. After incubation, the cells were washed twice, and then the stained cells from each patient were pooled. Finally, protein-specific B cells were gated as CD19⁺CD27⁺IgD^-RBD⁺S1⁺, CD19⁺CD27⁺IgD^-RBD⁺S1^- and CD19⁺CD27⁺IgD^-S1⁺RBD^- and sorted using a BD FACS Aria III (BD Biosciences, San Jose, CA, USA).

García-Vega M., Melgoza-González E.A., Hernández-Valenzuela S., Hinojosa-Trujillo D., Reséndiz-Sandoval M., Llamas-Covarrubias M.A., Loza-López M., Valenzuela O., Soto-Gaxiola A., Hernández-Oñate M.A., Mata-Haro V., Cassaniti I., Sammartino J.C., Ferrari A., Simonelli L., Pedotti M., Sun R., Zuo F., Baldanti F., Varani L., Marcotte H., Pan-Hammarström Q, & Hernández J. (2023). 19n01, a broadly neutralizing antibody against omicron BA.1, BA.2, BA.4/5, and other SARS-CoV-2 variants of concern. iScience, 26(4), 106562.

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Sorting SARS-CoV-2 RBD-Specific Plasma Cells

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Staining for sorting was performed using fresh lymph node single cell
suspensions in PBS supplemented with 2% FBS and 1mM EDTA (P2). Cells were
stained for 30 min on ice with biotinylated recombinant SARS-CoV-2 RBD diluted
in P2, washed twice, then stained for 30 min at 4°C with Fas-PE (Jo2, BD
Pharmingen), CD4-eFluor 780 (GK1.5, eBioscience), CD138-BV421 (281–2),
IgD-FITC (11–26c.2a), GL7-PerCP-Cy5.5, CD38-PE-Cy7 (90), CD19-APC (1D3),
and Zombie Aqua (all Biolegend) diluted in P2. Cells were washed twice and
single SARS-CoV-2 RBD-specific PBs (live singlet CD19⁺CD4⁻ IgD^lo Fas⁺ CD38^loCD138⁺ RBD⁺) and total PBs (live singlet
CD19⁺ CD4⁻ IgD^lo Fas⁺CD38^lo CD138⁺) were sorted using a FACSAria II into
96-well plates containing 2 μL Lysis Buffer (Clontech) supplemented with
1 U/μL RNase inhibitor (NEB) and immediately frozen on dry ice or bulk
sorted into PBS supplemented with 0.05% BSA and processed for single cell
RNAseq.

Alsoussi W.B., Turner J.S., Case J.B., Zhao H., Schmitz A.J., Zhou J.Q., Lei T., Rizk A.A., McIntire K.M., Chen R.E., Hassan A.O., Fox J.M., Winkler E.S., Thackray L.B., Kafai N.M., Amanat F., Krammer F., Watson C.T., Kleinstein S.H., Fremont D.H., Diamond M.S, & Ellebedy A.H. (2020). A potently neutralizing antibody protects mice against SARS-CoV-2 infection. Journal of immunology (Baltimore, Md. : 1950), 205(4), 915-922.

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Characterization of B Cell Subsets

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PFMCs were washed with PBS (4% FBS) and suspended at a concentration of 1x10 7 /200 µl, followed by staining with CD19-APC, IgD-FITC, CD27-PE-Cy7, CD38-APC-Cy7 (Biolegend, San Diego, CA, USA) for 30 min at 4˚C in the dark. Cells were then washed twice and re-suspended in 200 µl PBS (4% FBS). Within 2 h, the samples were acquired on a modified BD Canto II™ flow cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was performed using FlowJo software (Tree Star, Inc., Ashland OR, USA).
Statistical analysis. Statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). Differences in sex ratio of the two study cohorts were compared by Pearson's χ² test. Differences in age between the two study cohorts were evaluated by Student's t-test. Differences in BAFF level and the proportion of each B cell subset were evaluated by analysis of variance with Tukey's post hoc test for multiple comparisons. Correlations between two variables were analyzed by Spearman's analysis. P<0.05 was considered to indicate a statistically significant difference.

Wang X., Liang K.D., Zhang J.A., Liu G.B., Chen Z., Chen C., Zhuang Z.G., Liu Y.Q., Luo H.L., Li R.X., Zheng B.Y, & Xu J.F. (2018). Increased B cell activating factor is associated with B cell class switching in patients with tuberculous pleural effusion. Molecular medicine reports, 18(2).

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