Anti igd pe

Manufactured by BD

Sourced in United States

Anti-IgD-PE is a fluorescently labeled antibody that binds to the IgD receptor on the surface of cells. It is used in flow cytometry applications to detect and analyze cells expressing the IgD receptor.

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Anti-IgD (26 citations) IgD PE (18 citations) PE-labeled anti-human IgD (4 citations) PE-conjugated anti-IgD (4 citations) Anti-IgD-PE (IA6-2) (3 citations) Anti-IgD PE-Cy7 (2 citations) PE labeled anti-mouse IgD (2 citations) Anti-IgD-PE (clone 11-26c.2a); (2 citations)

12 protocols using anti igd pe

Activation of Human T Cells by IgD

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Human IgD was purchased from Abcam (Cambridge, MA, United States). CP-25 was provided by the Chemistry Lab of the Institute of Clinical Pharmacology of Anhui Medical University with a purity of 98.8% (Hefei, China). CP-25 was dissolved in DMEM. Herbimycin A (HA) was purchased from Millipore (Temecula, CA, United States). A770041 was purchased from Axon Medchem (Groningen, Netherlands). Biotinylated IgD was prepared in our laboratory using a protein biotinylation kit from Pierce Biotechnology (Rockford, IL, United States) according to the manufacturer’s instructions. Human CD4 microbeads were purchased from Miltenyi Biotec (Germany). PE-anti-CD69, PE-anti-CD154, PE-anti-CD62L, PE-anti-IgD, PE-cy5-anti-CD4 monoclonal antibodies (mAbs), APC-Cy7-streptavidin and isotype-matched PE-labeled mouse IgG2a mAbs were purchased from BD Pharmingen (San Diego, CA, United States). The anti-Lck antibody was purchased from Cell Signaling Technology (Danvers, MA, United States).

Wu Y.J., Chen H.S., Chen W.S., Dong J., Dong X.J., Dai X., Huang Q, & Wei W. (2018). CP-25 Attenuates the Activation of CD4+ T Cells Stimulated with Immunoglobulin D in Human. Frontiers in Pharmacology, 9, 4.

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Multiparameter Flow Cytometry of PBMCs

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Duplicate samples of human PBMCs (10⁶/sample) were stained with the following regents for 30 min at room temperature: fluorescein isothiocyanate conjugated anti-CD 4 (FITC-anti-CD4), phycoerythrin conjugated anti-CD8 (PE-anti-CD8), peridinin-chlorophyll-protein conjugated anti-CD3 mAb (PerCP-anti-CD3; Clone SK7/SK1/SK3; BD Tritest; San Jose, CA, USA), PE-anti-CD19, FITC-anti-CD138, PE-anti-IgD, FITC-anti-CD3, and PE-anti-CD16 + CD56+ (BD Bioscience; San Jose, CA, USA). Samples stained with FE-anti-IgG, FE-anti-IgG1, FE-IgG2a, and FITC-anti-IgG (BD Bioscience) were used as isotype controls. After staining, the cells were washed with PBS and analyzed using a FACSAria II flow cytometer (BD BioSciences; Franklin Lakes, NJ, USA). A minimum of 50,000 events per sample were analyzed using FlowJo software (v5.7.2) (FlowJo LLC; Ashland, OR, USA) [13 ].

Li C., Liu Y., Jiang Y., Xu N, & Lei J. (2017). Immunomodulatory constituents of human breast milk and immunity from bronchiolitis. Italian Journal of Pediatrics, 43, 8.

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Multi-parameter Immune Cell Profiling

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A total of 10⁶ PBMCs were plated in 96-well round-bottom plates in RPMI + 10% FBS (Gemini) + 1% penicillin, streptomycin, and l-glutamine (Invitrogen, Carlsbad, CA). After centrifugation and removal of media, cells were surface stained with 50 μL of a cocktail mix consisting of titrated volumes of LIVE/DEAD violet dye (Life Technologies, Grand Island, NY); anti-CD3, anti-CD14, and CD16 Pacific Blue; anti-CD19 PcP-Cy5.5; anti-CD27 APC; anti-IgD PE; anti-CD20 FITC; anti-CD24 PE-Cy7 (BD Biosciences, San Jose, CA); and anti-CD38 APC-AF700 (Beckman Coulter, Brea, CA) (table 2). A “dump” channel consisting of LIVE/DEAD dye and CD3, CD14, and CD16 Pacific Blue was used to exclude dead cells, T cells, and monocytes. After fixing the cells with 1% paraformaldehyde (PFA), they were acquired on a LSRII flow cytometer (BD Biosciences, San Jose, CA).

Guptill J.T., Yi J.S., Sanders D.B., Guidon A.C., Juel V.C., Massey J.M., Howard JF J.r., Scuderi F., Bartoccioni E., Evoli A, & Weinhold K.J. (2015). Characterization of B cells in muscle-specific kinase antibody myasthenia gravis. Neurology® Neuroimmunology & Neuroinflammation, 2(2), e77.

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Immune Cell Phenotyping by Flow Cytometry

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Splenocytes, thymocytes, and peritoneal cavity cells were resuspended in PBS containing 1% FBS and Rat Anti-Mouse CD16/CD32 Mouse BD Fc Block (BD Biosciences). The following antibody combinations were used: anti-CD4-PE (BD Biosciences, clone GK1.5) and anti-CD8a-PerCP (BD Biosciences, clone 53–6.7); anti-IgD-PE (BD Biosciences, clone 11-26c.2a) and anti-IgM-APC (SouthernBiotech, clone 1B4B1); anti-CD5-APC (BD Biosciences, clone 53–7.3) and anti-IgM-PE (SouthernBiotech, clone 1B4B1); and anti-CD21-APC (BD Biosciences, clone 7G6) and anti-CD23-FITC (BD Biosciences, clone B3B4). Flow cytometry was performed with a BD FACSCaliber and Flojo software (Treestar).

Yu J.W., Hoffman S., Beal A.M., Dykon A., Ringenberg M.A., Hughes A.C., Dare L., Anderson A.D., Finger J., Kasparcova V., Rickard D., Berger S.B., Ramanjulu J., Emery J.G., Gough P.J., Bertin J, & Foley K.P. (2015). MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses. PLoS ONE, 10(5), e0127083.

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Isolation and Sorting of Human B Cell Subsets

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About 50 mL of healthy human donor one (HD1) or healthy donor two (HD2) source leukocytes peripheral blood (Gulf Coast Regional Blood Center, Houston, TX, USA; E5318 product code) was used as a source for separating all nucleated cells from red blood cells using HetaSep (Stemcell Technologies) according to the manufacturer’s protocol. B cells were then extracted and enriched for from the nucleated cells using an EasySep Human CD19 + Positive Selection Kit (Stemcell Technologies) according to the manufacturer’s protocol. The enriched B cells were resuspended in ~500 µL of FACS buffer. These B cells were then stained with a 1:16 dilution of anti-IgD-PE (BD, cat#562024), a 1:16 dilution of anti-CD20-FITC (BD, cat#560962), a ~1:26 dilution of anti-CD19-v450 (BD, cat#560353), a 1:61 dilution of anti-CD3-PerCP-Cy5.5 (Biolegend, cat#300327), and a 1:16 dilution of anti-CD27⁻APC (BD, cat#558664) and then sorted for naive (CD20⁺CD19⁺IgD⁺CD3⁻CD27⁻) and memory (CD20⁺CD19⁺IgD⁻CD3⁻CD27⁺) cells on a FACSAria II.

Johnson E.L., Doria-Rose N.A., Gorman J., Bhiman J.N., Schramm C.A., Vu A.Q., Law W.H., Zhang B., Bekker V., Abdool Karim S.S., Ippolito G.C., Morris L., Moore P.L., Kwong P.D., Mascola J.R, & Georgiou G. (2018). Sequencing HIV-neutralizing antibody exons and introns reveals detailed aspects of lineage maturation. Nature Communications, 9, 4136.

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Immunofluorescence Analysis of Kidney and Spleen

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Kidneys and spleens of NZB/W mice were embedded in OCT compound (Sakura Finetek, Torrance, CA, USA) and snap-frozen in liquid nitrogen. Frozen sections were fixed in acetone and blocked with 10% normal donkey serum (Sigma Aldrich). Kidney sections were stained with a 1:200 dilution of anti-IgG-biotin (Sigma Aldrich), anti-IgM-biotin (Southern Biotech) and anti-C3-biotin (Bioss), followed by reaction with 1:200-dilution of streptavidin-cy3 (Invitrogen). Spleen sections were stained with anti-GL7-FITC, anti-CD4-APC, and anti-IgD-PE (all from BD Biosciences or eBioscience). Fluorescence images were acquired using a TCS SP5 confocal microscope (Leica). The area of GCs composed with GL7⁺ cells per image was calculated using ImageJ software (NIH, Bethesda, MD, USA).

Cho S., Jang E., Yoon T., Hwang H, & Youn J. (2022). A novel selective spleen tyrosine kinase inhibitor SKI-O-703 (cevidoplenib) ameliorates lupus nephritis and serum-induced arthritis in murine models. Clinical and Experimental Immunology, 211(1), 31-45.

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Phenotyping of B-cell Subsets in PBMCs

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PBMC samples were thawed from liquid nitrogen on the same day as the staining and 3x10⁶ PBMCs were used to study B-cell phenotype. PBMCs were stained with Live/Dead (Invitrogen), anti-IgM-PercP-Cy5.5 (BD), anti-IgD-PE (BD), anti-CD19-AlexaFluor780, anti-CD20-AlexaFluor700, anti-CD27-APC, anti-CD24-FITC, anti-CD38-PECy7 (all eBioscience) for 30min at 4°C. Cells were acquired on LSRFortessa (BD). Data was acquired until at least 5,000 events in the B-cell subset were counted. All flow cytometry data was analysed using FlowJo (Tree Star, Inc. Ashland, OR 97520 USA) or DIVA software (BD).

Nova-Lamperti E., Chana P., Mobillo P., Runglall M., Kamra Y., McGregor R., Lord G.M., Lechler R.I., Lombardi G, & Hernandez-Fuentes M.P. (2017). Increased CD40 ligation and reduced BCR signalling leads to higher IL-10 production in B-cells from tolerant kidney transplant patients. Transplantation, 101(3), 541-547.

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Basophil IgD and Galectin-9 Interactions

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Untouched human basophils were stripped of pre-bound endogenous IgD as described in published studies (Chen et al., 2009 (link)) and incubated in PBS with or without 50 μg/ml monoclonal IgD from human myeloma (Athens Research and Technology), 2.5 μg/ml galectin-9 (R&D Systems), or 50 μg/ml IgD premixed with 2.5 μg/ml galectin-9 on ice for 15 minutes. Following washing, basophils were sequentially stained with F(ab)2 to IgD biotin (Southern Biotech) and streptavidin (Life Technologies) along with antiFcɛRI FITC (clone: AER-37; eBioscience) and anti-CD44 PE (clone: IM7; Biolegend). Following additional washing, stained cells were fixed with 1 % paraformaldehyde, stained with 100 ng/ml DAPI (Sigma-Aldrich), and imaged. A similar strategy was used to image basophils included in splenocytes from Balb/c mice. These cells were incubated with an Fc-blocking 2.4G2 mAb (Tonbo Bioscience) and subsequently stained with anti-FcɛRI FITC (clone: MAR-1), anti-IgD PE (clone: 11–26c.2a) (BD Biosciences), anti-CD49b APC (clone: DX5) (Biolegend), and Ghost Violet 510 viability dye (Tonbo Bioscience). Following washing, stained cells were fixed with 1 % paraformaldehyde and stained with 5 μg/ml 7-AAD (Tonbo Bioscience).

Shan M., Carrillo J., Yeste A., Gutzeit C., Garzón D.S., Walland C., Pybus M., Grasset E.K., Yeiser A.J., Matthews D.B., van de Veen W., Comerma L., He B., Boonpiyathad T., Lee H., Blanco J., Osborne L.C., Siracusa M.C., Akdis M., Artis D., Mehandru S., Sampson H.A., Berin M.C., Chen K, & Cerutti A. (2018). Secreted IgD Amplifies Humoral T Helper-2 Responses by Binding Basophils Via Galectin-9 and CD44. Immunity, 49(4), 709-724.e8.

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Comprehensive B-cell Immunophenotyping

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B-cell subsets were analyzed on whole blood by incubating various combinations of monoclonal antibodies, including anti-human CD19 PerCP, anti-IgM APC, CD27 FITC, CD38 FITC, anti-IgD PE, CD21 PE, CD70 PE, CD27 APC, CD24 FITC, CD38 PE, CD183 PE (BD Pharmingen) and CD43 APC (Biolegend, San Diego, CA, USA) monoclonal antibodies, and corresponding isotypes. After staining, blood was lysed by 1 × lysing solution (BD Pharmingen) and washed with phosphate-buffered saline and analyzed. Flow cytometry was performed using FACSCalibur (Becton-Dickenson, San Jose, CA, USA) equipped with argon ion laser emitting at 488 nm (for FITC, PE and APC excitation). Forward and side scatters were used to gate and exclude cellular debris. Ten thousand cells were acquired and analyzed using the Flowjo software (Tree star Inc., Ashland, OR, USA).

Louis A.G., Yel L., Cao J.N., Agrawal S, & Gupta S. (2016). Common variable immunodeficiency associated with microdeletion of chromosome 1q42.1-q42.3 and inositol 1,4,5-trisphosphate kinase B (ITPKB) deficiency. Clinical & Translational Immunology, 5(1), e59-.

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Immunization and Analysis of Arthritis in Mice

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Flt3L^−/−, WT and Batf3^−/− mice were immunized as previously described [23 (link)] and were inspected three times a week for signs of arthritis by two independent observers. All mice were sacrificed on day 43 after CIA induction. Blood, LNs and paws were harvested for analysis. Single-cell suspensions were obtained and after erythrocyte lysis (red blood cell lysis buffer, 2 min at RT; Sigma), cells were stained with the indicated fluorochrome-conjugated antibodies for surface markers and intracellular cytokines. LN cells were stained using the following markers: anti-TNF (APC, eBioscience), anti-IL-2 (APC, eBioscience), anti-IL-17 (Alexa 488, eBioscience), anti-IFNγ (PercP Cy5.5, eBioscience), anti-IL10 (PE, eBioscience), anti-GM-CSF (PE, eBioscience), anti-B220 (PerCP, eBioscience), anti-CD19 (Alexa 700, eBioscience), anti-GL7 (biotin, eBioscience), anti-IgD (PE, BD Pharmingen), anti-CD38 (FITC, eBioscience), anti-CD95 (APC, eBioscience) and streptavidin (PE-Cy7, eBioscience). Synovial cells were isolated and stained using antibodies against CD11c (PE-Cy7, eBioscience), MHCII (APC-Cy7, eBioscience), CD11b (Alexa 700) and CD103 (FITC, eBioscience). Serum levels of antibodies against chicken collagen type II (cCII) were measured by ELISA. Further details are described in supplementary methods.

Ramos M.I., Garcia S., Helder B., Aarrass S., Reedquist K.A., Jacobsen S.E., Tak P.P, & Lebre M.C. (2020). cDC1 are required for the initiation of collagen-induced arthritis. Journal of Translational Autoimmunity, 3, 100066.

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