B220 bv510

Manufactured by BioLegend

Sourced in United States

B220-BV510 is a fluorochrome-conjugated antibody that binds to the B220 antigen, also known as CD45R, which is expressed on the surface of B cells. This product is designed for use in flow cytometry applications.

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B220 on BV510 Anti-mouse B220-BV510 BV510 anti-B220

5 protocols using b220 bv510

Multiparameter Flow Cytometry Analysis

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Spleens were mechanically disrupted, single-cell suspensions were generated, and red blood cells were lysed with ammonium chloride TRIS. Cells were resuspended in PBS containing 1% FBS and incubated with indicated antibodies. For analysis of cell surface markers, antibodies against the following molecules were used: B220-PE (RA3–6B2; BioLegend), B220-BV786 (RA3–6B2; BD Biosciences), B220-BV510 (RA3–6B2; BioLegend), CD4-BV711 (GK1.5; BioLegend), CD8 BV421 (53–6.7; BioLegend), CD138-PECy7 (281–2; BioLegend), CD69-BV786 (H1.2F3; BD Biosciences), CD86-PerCPCy5.5 (GL-1; BioLegend). After cell surface staining, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD) (as per manufacturer’s instructions) and stained with Dylight650-E4 anti-Ars/A1 idiotype (produced and conjugated in our laboratory) OR HEL-650 (produced and conjugated in our laboratory) and Alexa Fluor 488-anti-GFP (rabbit polyclonal; Life Technologies). Events were collected on a CyAn ADP (Dako) and subsequent analysis using FlowJo software (Tree Star).

Franks S.E., Getahun A, & Cambier J.C. (2019). A Precision B cell-targeted Therapeutic Approach to Autoimmunity Caused by PI3K Pathway Dysregulation. Journal of immunology (Baltimore, Md. : 1950), 202(12), 3381-3393.

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Comprehensive FACS analysis of murine blood

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50 µl of blood was 1:1 diluted in PBS; lymphocytes were separated using 100 µl Lymphocyte Separation Medium 1077 (C-44010, PromoCell, Heidelberg, Germany). Cells were stained for 30 min/4 °C with: CD4-PE/Cy5 (1:1000; 130312), CD8a-PE/Cy7 (1:500; 100722), B220-BV510 (1:300; 103248), Gr-1-PE (1:1000; 108408), CD11b-PerCP/Cy5.5 (1:1000; 101227), F4/80-FITC (1:1000; 123107, all from BioLegend). Filtered samples were acquired on FACSAria Sorp (BD-Biosciences), and data analyzed by FlowJo software (BD-Biosciences).

Pan H., Steixner-Kumar A.A., Seelbach A., Deutsch N., Ronnenberg A., Tapken D., von Ahsen N., Mitjans M., Worthmann H., Trippe R., Klein-Schmidt C., Schopf N., Rentzsch K., Begemann M., Wienands J., Stöcker W., Weissenborn K., Hollmann M., Nave K.A., Lühder F, & Ehrenreich H. (2020). Multiple inducers and novel roles of autoantibodies against the obligatory NMDAR subunit NR1: a translational study from chronic life stress to brain injury. Molecular Psychiatry, 26(6), 2471-2482.

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Tetramer-based Identification of Antigen-specific CD8+ T Cells

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Draining LNs were processed by glass slide maceration 7 days after injection, washed, and suspended in FACS (2% FBS in PBS) buffer containing Tetramer (SIINFEKL)-PE (1:400) (NIH tetramer core facility), CD8 APC-Cy7 (Biolegend clone 53-6.7 1:400) for 1 hr at 37C. Cells were washed and stained for 30 min in CD44 PerCP Cy5.5 (Biolegend clone IM7, 1:400), B220 BV510 (Biolegend clone RA3-6B2, 1:300). Samples were ran on the FACS Canto II flow cytometer (BD).

Walsh S.M., Sheridan R.M., Lucas E.D., Doan T.A., Ware B.C., Schafer J., Fu R., Burchill M.A., Hesselberth J.R, & Tamburini B.A. (2021). Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node. eLife, 10, e62781.

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Multicolor Flow Cytometry of Heart Cells

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Cytofluorimetric analysis was performed as previously described [29 (link),30 (link)]. Briefly, hearts were collected and cut into small pieces with a blade, and then incubated with collagenase type IV for 1 h and 30′ at 37 °C with agitation. The obtained cell suspension was passed through a 70 μm and then a 40 μm cell strainer; the cells were then counted on a hemacytometer, collected by centrifugation at 1200 rpm, and suspended in 200 μL of calcium/magnesium-free PBS (phosphate-buffered saline) with 2% FBS (foetal bovine serum). They were then divided into two tubes for the staining. The cells were then incubated on ice for 30 min with the following antibodies: CD45 PE/Cy7, F4/80 APC, Ly6g PE Fluor 610, CD11b APC/Cy7, CD206 PERCP/Cy5.5 Ly6c BV-510, I-Ab FITC (tube 1) and CD45 PE/Cy7, CD3 PERCP Cy5.5, B220 BV-510, CD4 AF488, and CD8 PE (tube 2), all by Biolegend, (San Diego, CA, USA). Cells were then washed with 3 mL of calcium/magnesium-free PBS and resuspended in 200 μL of calcium/magnesium-free PBS with 2% FBS. Samples were acquired with a CyAn ADP (Agilent DAKO, Santa Clara, CA, USA), and acquired data were analysed using FlowJo software version 10.1.

Morroni J., Schirone L., Valenti V., Zwergel C., Riera C.S., Valente S., Vecchio D., Schiavon S., Ragno R., Mai A., Sciarretta S., Lozanoska-Ochser B, & Bouchè M. (2022). Inhibition of PKCθ Improves Dystrophic Heart Phenotype and Function in a Novel Model of DMD Cardiomyopathy. International Journal of Molecular Sciences, 23(4), 2256.

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Multiparameter Flow Cytometry Profiling

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After resuspension of mechanically dissociated cells in fluorescence-activated cell sorting (FACS) buffer (pH = 7.4, 0.1M PBS, 1 mM EDTA, 1% BSA), the cells underwent staining with conjugated antibodies/dyes. Conjugated antibodies were diluted 1:200 in FACS buffer, and the following antibodies and dyes were used: Ghost UV450 (Tonbo Biosciences, Catalog #: 13-0868-T500) to visualize live/dead cells; The copyright holder for this preprint this version posted October 9, 2020. ; https://doi.org/10.1101/2020.10.08.331801 doi: bioRxiv preprint (Biolegend, Catalog #: 100721); CD4 BUV496 to visualize T helper cells (BD Bioscience, Catalog #: 564667); B220 BV510 to visualize B cells (Biolegend, Catalog #: 103247); and PDL-1 PE-Cy7 to visualize the tolerogenic ligand PDL-1 (Biolegend, Catalog #: 124313). After staining for 30 minutes at 4ºC, cells were washed three times in FACS buffer and processed using a BD LSR II. For the CD45 IV timing experiments, cells were processed with Cytek's 3-laser Northern Lights.

Hsu M., Madrid A., Choi Y.H., Laaker C., Herbáth M., Sándor M., & Fábry Z. (2020). Neuroinflammation alters the phenotype of lymphangiogenic vessels near the cribriform plate.

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