Igd pe cy7

Manufactured by BD

Sourced in Netherlands

IgD-PE-Cy7 is a fluorescently labeled antibody used for the detection and analysis of IgD-positive cells in flow cytometry applications. The PE-Cy7 dye provides a combination of brightness and spectral properties that allow for multicolor flow cytometry analysis.

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Lab products found in correlation

Cd14 bv510, by BioLegend (5 mentions) Cd3 bv510, by BioLegend (5 mentions) Facsaria 2, by BD (4 mentions) Cd56 bv510, by BioLegend (4 mentions) Cd27 bv605, by BioLegend (3 mentions) Cd21 bv711, by BD (3 mentions) Cd19 ecd, by Beckman Coulter (3 mentions) Anti human cd19 alexa 700, by BioLegend (2 mentions) Cd3 percp cy5, by BD (2 mentions) Calcien, by Thermo Fisher Scientific (2 mentions) Macs human b cell isolation kit, by Miltenyi Biotec (2 mentions) Live dead violet, by Thermo Fisher Scientific (2 mentions) Cd20 af700, by BD (2 mentions) Igm fitc, by BD (2 mentions) Cd8 bv510, by BioLegend (2 mentions) Cd19 bv750, by BioLegend (2 mentions) Live dead blue, by Thermo Fisher Scientific (2 mentions) Iga dylight 405, by Jackson ImmunoResearch (2 mentions) Igm brilliant ultraviolet buv 395, by BD (2 mentions) Igg allophycocyanin apc h7, by BD (2 mentions)

Close spelling variants of this product

IgD PE (18 citations) Anti-IgD PE-Cy7 (2 citations)

11 protocols using igd pe cy7

SARS-CoV-2 Spike Protein Binding Assay

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Naive B cells were purified from PBMCs using the MACS Human B Cell isolation kit (Miltenyi Biotec) and incubated with 25nM of each SARS-CoV-2 probe (RBD-mFc-BV650, ΔRBM-mFc-BV786, spike-PE, and spike-APC) for 30 min at 4°C. Cells were stained with anti-human CD19 (Alexa-700; BioLegend, cat. no 302226), CD3 (PerCP-Cy5; BD Biosciences, cat no. 560835), IgD (PE-Cy7; BD Biosciences, cat no. 561314), IgG (BV711; BD Biosciences, cat no. 740796), CD27 (BV510; BD Biosciences, cat no. 750167), LiveDead Violet (Invitrogen), and Calcien (Invitrogen) for an additional 30 min. RBM-specific naive B cells, defined as CD19⁺/CD3⁻/IgG⁻/IgD⁺/spike PE⁺/spike APC⁺/RBD⁺/ΔRBM⁻⁻, were single-cell sorted using BD FACS Aria II (BD Biosciences) into 96-well plates containing lysis buffer supplemented with 1% BME. Within the CD19⁺/IgG⁻/IgD⁺ gated cells, we also confirmed that 97% of the events were CD27 negative. Plates were stored at −80 °C for subsequent analysis. Flow cytometry data was analyzed using FlowJo software version 10.7.1.

Feldman J., Bals J., Altomare C.G., St. Denis K., Lam E.C., Hauser B.M., Ronsard L., Sangesland M., Moreno T.B., Okonkwo V., Hartojo N., Balazs A.B., Bajic G., Lingwood D, & Schmidt A.G. (2021). Naive human B cells engage the receptor binding domain of SARS-CoV-2, variants of concern, and related sarbecoviruses. Science immunology, 6(66), eabl5842.

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Isolation and Sequencing of IgG+ Memory B Cells

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B cells were isolated from PBMCs using CD19 microbeads (Miltenyi Biotec) and stained with DAPI (Thermo Fisher), CD20-AF 700, IgG-APC, IgD-Pe-Cy7, IgM-FITC, and CD27-PE (all BD Biosciences) for 30 min on ice. 200,000 CD20⁺IgG⁺IgM^-IgD^-CD27^- B cells were sorted into FBS (Sigma-Aldrich) using a BD FACSAria Fusion, and RNA of sorted B cells was isolated with the RNeasy Micro Kit (QIAGEN). cDNA was generated by template-switch reverse transcription according to the SMARTer RACE 5′/3′ manual using the SMARTScribe Reverse Transcriptase (Takara) with a template-switch oligo including an 18-nucleotide unique molecular identifier. Heavy-chain variable regions were amplified with an IgG-specific nested PCR and amplicons were used for library preparation and MiSeq 2x300 bp sequencing (Illumina). Raw NGS reads were pre-processed and assembled to final sequences as previously described (Ehrhardt et al., 2019 (link)).

Schommers P., Gruell H., Abernathy M.E., Tran M.K., Dingens A.S., Gristick H.B., Barnes C.O., Schoofs T., Schlotz M., Vanshylla K., Kreer C., Weiland D., Holtick U., Scheid C., Valter M.M., van Gils M.J., Sanders R.W., Vehreschild J.J., Cornely O.A., Lehmann C., Fätkenheuer G., Seaman M.S., Bloom J.D., Bjorkman P.J, & Klein F. (2020). Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody. Cell, 180(3), 471-489.e22.

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Phenotyping PfCSP+ Memory B Cells

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Cryopreserved PBMCs from the VRC 314 trial were thawed and stained for 30 minutes at room temperature in the dark with the following panel: 1:500 Blue LIVE/DEAD (Thermo Fisher Scientific L23105), 18 μg/mL IgA-Dylight 405 (Jackson Immunoresearch 109-475-011), 1:50 CD14-BV510 (BioLegend 301842), 1:50 CD3-BV510 (BioLegend 317332), 1:100 CD8-BV510 (301048), 1:50 CD56-BV510 (BioLegend 318340), 1:50 CD27-BV605 (BioLegend 302830), 1:40 CD21-BV711 (BD Biosciences 563163), 1:50 CD19-BV750 (BioLegend 302236), 1:125 IgD-PECy7 (BD Biosciences 561314), 1:20 IgM-Brilliant Ultraviolet (BUV)395 (BD Biosciences 563903), 1:125 CD38-Alexa Fluor 700 (BD Biosciences 560676), 1:40 IgG-allophycocyanin (APC)H7 (BD Biosciences 561297), 1:25 PfCSP-Brilliant Blue (BB)660, and 1:25 PfCSP-BUV737. The PfCSP probes were made by conjugating biotinylated PfCSP to streptavidin tagged with the relevant fluorescent dye. The cells were sorted using a BD FACS Aria II and analyzed with FlowJo software (Tree Star). Cells were gated on live CD19⁺CD14^-CD3^-CD8^-CD56^-CD21⁺CD27⁺IgA⁺/IgG⁺, with CD27⁺⁺CD38⁺⁺ cells excluded.

Tan J., Cho H., Pholcharee T., Pereira L.S., Doumbo S., Doumtabe D., Flynn B.J., Schön A., Kanatani S., Aylor S.O., Oyen D., Vistein R., Wang L., Dillon M., Skinner J., Peterson M., Li S., Idris A.H., Molina-Cruz A., Zhao M., Olano L.R., Lee P.J., Roth A., Sinnis P., Barillas-Mury C., Kayentao K., Ongoiba A., Francica J.R., Traore B., Wilson I.A., Seder R.A, & Crompton P.D. (2021). Functional human IgA targets a conserved site on malaria sporozoites. Science translational medicine, 13(599), eabg2344.

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SARS-CoV-2 Spike and RBD Staining of PBMCs

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PBMCs were stained with SARS-CoV-2 Spike and RBD probes, as previously described (Juno et al., 2020 (link)). Probes were generated by sequential addition of streptavidin-phycoerythrin (PE) (Thermo Fisher Scientific) to trimeric S protein biotinylated using recombinant Bir-A (Avidity), while SARS-CoV-2 RBD was labeled to APC using an APC Conjugation Lightning-Link Kit (Abcam). PBMCs were surface stained with Aqua viability dye (Thermo Fisher) and monoclonal antibodies against CD19-ECD (#IM2708U, Beckman Coulter), IgM BUV395 (#563903), CD21 BUV737 (#564437), IgD PE-Cy7 (#561314), IgG BV786 (#564230), streptavidin-BV510 (#563261) (BD Biosciences), CD20 APC-Cy7 (#302314), CD14 BV510 (#301841), CD3 BV510 (#317332), CD8a BV510 (#301048), CD16 BV510 (#302048), CD10 BV510 (#312220) and CD27 BV605 (#302829) (BioLegend). Cells were washed, fixed with 1% formaldehyde and acquired on a BD LSRII Fortessa.

Nguyen T.H., Rowntree L.C., Petersen J., Chua B.Y., Hensen L., Kedzierski L., van de Sandt C.E., Chaurasia P., Tan H.X., Habel J.R., Zhang W., Allen L.F., Earnest L., Mak K.Y., Juno J.A., Wragg K., Mordant F.L., Amanat F., Krammer F., Mifsud N.A., Doolan D.L., Flanagan K.L., Sonda S., Kaur J., Wakim L.M., Westall G.P., James F., Mouhtouris E., Gordon C.L., Holmes N.E., Smibert O.C., Trubiano J.A., Cheng A.C., Harcourt P., Clifton P., Crawford J.C., Thomas P.G., Wheatley A.K., Kent S.J., Rossjohn J., Torresi J, & Kedzierska K. (2021). CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity. Immunity, 54(5), 1066-1082.e5.

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SARS-CoV-2 Spike Protein Binding Assay

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Immunophenotyping Immortalized B Cells

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Mock- or MCM-treated immortalized B cells or TAB were stained with the following antibodies or matched isotypes and analyzed on a FACS Aria III (BD): CD19 BV711 (clone SJ25C1, 0.06 µg/100 µl, catalog number 563036), CD20 AF700 (clone 2H7, 0.5 µg/100 µl, 560631), CD24 PE-CF594 (clone ML5, 1 µg/100 µl, 562405), CD27 BV421 (clone M-T271, 0.25 µg/100 µl, 562513), CD38 APC (clone HIT2, 0.125 µg/100 µl, 555462), CD138 PE (clone MI15, 0.125 µg/100 µl, 552026), IgD PE-Cy7 (clone IA6-2, 0.125 µg/100 µl, 561314), IgG FITC (clone G18-145, 0.125 µg/100 µl, 555786), IgM BV605 (clone G20-127, 0.5 µg/100 µl, 562977) (all BD biosciences). Live/dead cell exclusion was performed by addition of 7-AAD (5 µg/ml, Calbiochem) prior to acquisition of the samples. Data were analyzed using FlowJo 10.4.2 (FlowJo LLC). The gating strategy is shown in Supplementary Fig. 10.

Griss J., Bauer W., Wagner C., Simon M., Chen M., Grabmeier-Pfistershammer K., Maurer-Granofszky M., Roka F., Penz T., Bock C., Zhang G., Herlyn M., Glatz K., Läubli H., Mertz K.D., Petzelbauer P., Wiesner T., Hartl M., Pickl W.F., Somasundaram R., Steinberger P, & Wagner S.N. (2019). B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma. Nature Communications, 10, 4186.

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Comprehensive B Cell Immunophenotyping

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LN tissue was put through a 70 μm (BD Falcon, San Jose, CA) cell strainer to obtain a single cell suspension. Cells were washed with PBS containing 0.01% NaN₃ and 0.5% BSA and stained for 30 min at 4°C with directly labelled antibodies: CD45 PercP-Cy5.5, CD19 Alexa-700, CD27 APC-H7, IgD Pe-Cy7, CD20 PE, IgM FITC, CD69 PE, CD69 PerCP, CD21 APC, CD23 PE, CD25 APC, CD267 PE, BAFF-R FITC, CD16 Percp-Cy5.5, CD56 PE, CD55 PE, CD59 FITC (BD Biosciences, Breda, the Netherlands), HLA-DR Alexa-700 (eBioscience, Vienna, Austria), CD3 FITC (Sanquin, Amsterdam, the Netherlands). After incubation cells were washed and immediately analysed on a FACS CANTO II (BD Biosciences). To enable the measurement of different B cell subsets, a seven-colour FACS panel was set-up using antibodies against CD19, IgD, IgM, CD27, CD21, CD23 and CD45 (for normalization). Data were analysed using FlowJo software (Treestar, Ashland, OR, USA) and presented as frequencies, absolute numbers relative to 100 000 CD45⁺ lymphocytes or geometric mean fluorescence intensity (normalized on negative populations).

Ramwadhdoebe T.H., van Baarsen L.G., Boumans M.J., Bruijnen S.T., Safy M., Berger F.H., Semmelink J.F., van der Laken C.J., Gerlag D.M., Thurlings R.M, & Tak P.P. (2019). Effect of rituximab treatment on T and B cell subsets in lymph node biopsies of patients with rheumatoid arthritis. Rheumatology (Oxford, England), 58(6), 1075-1085.

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Isolation of SARS-CoV-2 Memory B Cells

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Cryopreserved PBMCs from COVID-19 convalescent donors were thawed and stained with DAPI (BD564907), CD14-BV510 (BioLegend 301842), CD3-BV510 (BioLegend 317332), CD56-BV510 (BioLegend 318340), CD19-ECD (Beckman Coulter IM2708U), CD21-BV711 (563163), IgA-Alexa Fluor 647 (Jackson Immunoresearch 109-606-011), IgD-PE-Cy7 (BD 561314), IgM-PerCP-Cy5.5 (BD561285), CD27-Alexa Fluor 488 (BioLegend 393204) and CD38-APC-Cy7 (BioLegend 303534). Stained cells were then sorted using the BD FACSAria IIIu in a BSL3 facility. This procedure involved gating out all but live CD19⁺CD14^-CD3^-CD56^-IgM^-IgD^- cells, and then gating on IgA to yield purified populations of IgA-producing and IgG-producing memory B cells (MBCs).

Dacon C., Peng L., Lin T.H., Tucker C., Lee C.C., Cong Y., Wang L., Purser L., Cooper A.J., Williams J.K., Pyo C.W., Yuan M., Kosik I., Hu Z., Zhao M., Mohan D., Peterson M., Skinner J., Dixit S., Kollins E., Huzella L., Perry D., Byrum R., Lembirik S., Murphy M., Zhang Y., Yang E.S., Chen M., Leung K., Weinberg R.S., Pegu A., Geraghty D.E., Davidson E., Doranz B.J., Douagi I., Moir S., Yewdell J.W., Schmaljohn C., Crompton P.D., Mascola J.R., Holbrook M.R., Nemazee D., Wilson I.A, & Tan J. (2023). Rare, convergent antibodies targeting the stem helix broadly neutralize diverse betacoronaviruses. Cell Host & Microbe, 31(1), 97-111.e12.

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Phenotyping Memory B Cells by Flow Cytometry

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Cryopreserved PBMCs were thawed and stained with the following panel: DAPI (BD564907), CD14-BV510 (BioLegend, 301842), CD3-BV510 (BioLegend, 317332), CD56-BV510 (BioLegend, 318340), CD19-ECD (Beckman Coulter, IM2708U), CD21-BV711 (BD, 563163), IgA-Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-011), IgD-PE-Cy7 (BD, 561314), and IgM-PerCP-Cy5.5 (BD, 561285), CD27-Alexa Fluor 488 (BioLegend, 393204) and CD38-APC-Cy7 (BioLegend, 303534). The cells were sorted using the BD FACSAria IIIu in a BSL3 facility and gated on live CD19⁺CD14⁻CD3⁻CD56⁻IgM⁻IgD⁻IgA⁺/IgA⁻ (memory B cells).

Dacon C., Tucker C., Peng L., Lee C.C., Lin T.H., Yuan M., Cong Y., Wang L., Purser L., Williams J.K., Pyo C.W., Kosik I., Hu Z., Zhao M., Mohan D., Cooper A., Peterson M., Skinner J., Dixit S., Kollins E., Huzella L., Perry D., Byrum R., Lembirik S., Zhang Y., Yang E.S., Chen M., Leung K., Weinberg R.S., Pegu A., Geraghty D.E., Davidson E., Douagi I., Moir S., Yewdell J.W., Schmaljohn C., Crompton P.D., Holbrook M.R., Nemazee D., Mascola J.R., Wilson I.A, & Tan J. (2022). Broadly neutralizing antibodies target the coronavirus fusion peptide. bioRxiv.

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Isolation and Phenotyping of Plasma Cells

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At 3 days post-infection cells were collected and pelleted at 1400 rpm for 3 minutes into 12x75mm round bottom tubes. Cells were resuspended in 200μl PBS containing (0.4ng/ml) fixable viability stain (BD 565388) and incubated on ice for 15 minutes. Cells were pelleted and resuspended in 200μl cold MACS buffer containing 5% FBS (Sort Block) and incubated on ice for 10 minutes after which 200μl cold MACS buffer was added. Cells were pelleted and resuspended in MACS buffer containing B cell phenotype panel as follows for 15 minutes on ice: (volumes indicated are for each 1(10)^6 cells and were scaled depending upon the number of cells being stained for sorting). For single cell sorting of plasma cells the panel was follows: CD19-PerCPCy5.5 (5μl, BD 561295), CD20-PE-Cy7 (5ul, BD 560735), and CD138-APC (5μl, Biolegend 352307). For other lineages the panel was as follows: CD19-PerCPCy5.5 (5μl, BD 561295), CD38-APC (20μl, BD 555462), IgD-PE-Cy7 (5, BD 561314), CD27-PE-Cy5 (5μl eBioscience 15-0279-42). After incubation, 500μl MACS buffer was added and pelleted lymphocytes were washed with a further 500μl of MACS buffer prior to being resuspended in 200μl MACS buffer and put through a cell strainer before sorting using the 70-micron nozzle on a BD FACSAria Fusion Cell Sorter.

Aalam F., Nabiee R., Castano J.R, & Totonchy J. (2020). Analysis of KSHV B lymphocyte lineage tropism in human tonsil reveals efficient infection of CD138+ plasma cells. PLoS Pathogens, 16(10), e1008968.

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