Igd apc

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IgD-APC is a monoclonal antibody labeled with the fluorescent dye Allophycocyanin (APC). It is designed to detect and quantify the expression of the immunoglobulin D (IgD) molecule on the surface of cells.

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IgD APC-Cy7 (11 citations) IgD-APC-Cy7 clone 11–26c2a (3 citations) APC-conjugated mouse antihuman IgD (2 citations) Anti-mouse IgD APC-Cy7 (2 citations)

4 protocols using igd apc

Comprehensive B Cell Immunophenotyping

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Flow cytometric analysis was performed on BD® LSR II Flow Cytometer (Marshallscientific, USA), and data were analyzed with FlowJo10.0 software (Treestar, Ashland, OR, USA). Anti- mouse B220-FITC (103205, Biolegend, USA), CD43-PE-Cy7 (143210, Biolegend, USA), CD24-PE (101807, Biolegend, USA), CD19-PE-Cy7 (552854, BD, USA), CD19-BV421 (115520, Biolegend, USA), CD19-PE (115508, Biolegend, USA), CD69-FITC (104506, Biolegend, USA), CD5-PE (100608, Biolegend, USA), CD86-APC (105011, Biolegend, USA), CD95-PE (152608, Biolegend, USA), BP-1-Alexa Fluor 647 (108312, Biolegend, USA), IgD-APC (405714, Biolegend, USA), IgM-PE (406507, Biolegend, USA), and corresponding isotype control antibodies were purchased from Biolegend. Antibodies were listed in Supplementary Table 3.

Kang X., Chen S., Pan L., Liang X., Lu D., Chen H., Li Y., Liu C., Ge M., Zhang Q., Liu Q, & Xu Y. (2022). Deletion of Mettl3 at the Pro-B Stage Marginally Affects B Cell Development and Profibrogenic Activity of B Cells in Liver Fibrosis. Journal of Immunology Research, 2022, 8118577.

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Profiling Memory B Cells from Vaccination

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To quantify and profile memory B cells from vaccination, single‐cell suspensions were prepared from inguinal lymph nodes, as previously described.³² Cells were stained with Zombie live/dead (Biolegend, San Diego, CA, USA), FcR‐blocked (anti‐CD16/CD32, BD Bioscience, Franklin Lakes, NJ, USA) and stained with a cocktail containing anti‐mouse B220‐PECy7, CD38‐PE, IgD‐APC, CD95 (Fas)‐BV605, IgM‐FITC, IgG‐APCCy7 (all Biolegend), for 30 min on ice in FACS buffer (PBS, 1% FBS, 0.5% NaN₃). Cells were finally fixed with 100 µL of 4% PFA for 20 min on ice. B memory cells were defined as B220⁺ CD38^+/− Fas^low IgD^‐ IgM⁺ or IgG⁺. Samples were acquired by flow cytometry on a FACS Attune (Invitrogen) and analysed with FlowJo software (BD). Representative FACS plots are shown in Supplementary figure 2a.

Kavian N., Hachim A., Cowling B.J, & Valkenburg S.A. (2021). Repeated influenza vaccination provides cumulative protection from distinct H3N2 viruses. Clinical & Translational Immunology, 10(6), e1297.

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Isolation and Characterization of Murine Bone Marrow B Cells

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Bone marrow was obtained from the mouse tibia and femur as previously demonstrated [12 (link)]. Briefly, each degloved leg was cleaned with a razor to remove all muscle tissue. The bone marrow was flushed with 10 mL RPMI 1640 1× media (Mediatech) supplemented with 5% heat-inactivated defined fetal bovine serum (FBS) (Hyclone), 2 mM L-glutamine, and 1% penicillin/streptomycin and placed into 20 mL of the same media. Once the bone marrow was filtered and the red blood cells lysed, 0.5 × 10⁶ cells were transferred to each well of a 96 well plate. The bone marrow was stained for 10 minutes on ice with FcR Block (Miltenyi Biotec) followed by 20 minutes with an antibody cocktail containing B220-FITC (Miltenyi Biotec), CD19-PerCP/Cy5.5 (Biolegend), IgM-PE (Southern Biotech), IgD-APC (Biolegend) and CD21-APC/Cy7 (Biolegend) in 1× PBS supplemented with 0.1% BSA. Dead cells were stained with Sytox Blue (Invitrogen).

Teague H., Harris M., Whelan J., Comstock S.S., Fenton J.I, & Shaikh S.R. (2015). Short-term consumption of n-3 PUFAs increases murine IL-5 levels but IL-5 is not the mechanistic link between n-3 fatty acids and changes in B cell populations. The Journal of nutritional biochemistry, 28, 30-36.

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Splenic B Cell Subset Isolation

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Spleens were mechanically disrupted and red blood cells were osmotically lysed to generate a single cell suspension [13 (link)]. B220⁺ B cells were purified from splenocytes with a negative selection microbead kit obtained from Miltenyi Biotec. 0.5 × 10⁶ splenic B cells were transferred to a 96 well plate and stained for 20 minutes on ice with a combination of B220-FITC (Miltenyi Biotec), IgM-PE (Southern Biotech), IgD-APC (Biolegend), CD19-PerCP/Cy5.5 (Biolegend), CD40-PerCP/Cy5.5 (Biolegend), CD21-APC/Cy7 (Biolegend), and MHC class II-PE (Bio X Cell) in 1× PBS supplemented with 0.1% BSA. The splenic B cell subsets analyzed were IgM⁺IgD^loCD21^lo, IgM⁺IgD^hiCD21^lo, IgM⁺IgD^hiCD21^hi, and IgM⁺IgD^loCD21^hi [12 (link), 19 (link)].

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