B220 pe cy7

Manufactured by Cytek Biosciences

The B220-PE-Cy7 is a fluorochrome-conjugated antibody that targets the B220 antigen, also known as the CD45R surface marker, on B lymphocytes. It is designed for use in flow cytometry applications to identify and quantify B cell populations within a sample.

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3 protocols using b220 pe cy7

Immunophenotyping Murine Immune Cells

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Cells were resuspended at a concentration of 10⁶/100 μl in FACS buffer (1x PBS, EDTA, 1% BSA) and first stained for 15 min on ice with Fc block (BD Biosciences, #553142). Next, antibody cocktails were added for a further 15 min on ice. Following staining, cells were washed with 10 volumes 1× with FACS buffer. The following antibodies were used in this study and are from BD Biosciences: CD138⁻BV711 (#563193, Clone 281-2, 1:200); and Tonbo Biosciences: B220-PE-Cy7 (#60-0452, Clone RA3-6B2, 1:400), CD11b-FITC (#35-0112, Clone M1/70, 1:100), Viability Ghost Dye-APC-Cy7 (#13-0865-T100, 1:100). Analysis was performed on a LSRII and sorting was performed on an ARIAII instrument (BD Biosciences).

Scharer C.D., Barwick B.G., Guo M., Bally A.P, & Boss J.M. (2018). Plasma cell differentiation is controlled by multiple cell division-coupled epigenetic programs. Nature Communications, 9, 1698.

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Multicolor Flow Cytometry Protocol for Murine Immune Cell Analysis

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PB was collected from the retro-orbital plexus in heparinized capillary tubes (Fisherbrand, Pittsburgh, PA) and lysed in red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO). Cells were stained with the following antibodies: CD45.1-APC (A20) (Biolegend, San Diego, CA) or CD45.1-FITC (A20) (BD Biosciences, San Diego, CA), B220-PECy7 and CD8-PECy7 (53–6.7) (Tonbo Biosciences, San Diego, CA), CD45.2-V500 (104), B220- PerCPCy5.5 (RA3-6B2), Gr1-PerCPCy5.5 (RB6-8C5), Cd11b-PerCPCy5.5 (M1/70) and CD4-PECy7 (RM4-5) (BD Biosciences, San Diego, CA). All antibodies were used at 1:200 dilution. 4’,6-diamidino-2-phenylindole (DAPI) staining was used to gate live events. Analysis was performed on a LSR Fortessa and a BD FACSAria III SORP (Special Order Research Product, which contains the following LASERs: 405 nm, 445 nm, 488 nm, 562 nm, and 640 nm) (both BD Biosciences, San Diego, CA) and the data analyzed with FlowJo version 9.4.11 (Tree Star, Ashland, OR). To discern the four Confetti colors: the filter arrangements for each LASER were: CFP, 445 nm LASER—470/24 band-pass filter (BP); GFP, 488 nm LASER—515/20 BP and 505 long-pass filter (LP); YFP, 488 nm LASER—545/10 BP and 525 LP; RFP, 562 nm LASER—610/20 BP and 600 LP^{48 (link)}.

Ganuza M., Chabot A., Tang X., Bi W., Natarajan S., Carter R., Gawad C., Kang G., Cheng Y, & McKinney-Freeman S. (2018). Murine hematopoietic stem cell activity is derived from pre-circulation embryos but not yolk sacs. Nature Communications, 9, 5405.

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Detailed Flow Cytometry for B Cell Subsets

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Cells were resuspended at 10⁶ per 100μl of FACS buffer (1X PBS, 1%BSA, and 2mM EDTA) and blocked with anti-Fc (anti-CD16/CD32) (Tonbo Biosciences, 2.4G2). The following antibodies were used to for staining: B220-PE-Cy7 (Tonbo Biosciences RA3-6B2), CD138-BV711 (BD, 281–2), GL7 eFluor660 (eBioscience, GL-7), CD11b-FITC (Tonbo Biosciences, M1/70), and Ghost Dye^™ Red 780 (Tonbo Biosciences 13–0865) to asses viability. The Annexin V FITC Apoptosis Detection Kit (eBioscience BMS500FI-100) was used to assess cell death. Cells were stained for 40 min and fixed using 1% paraformaldehyde.
Enrichment of CD138+ PB was performed by staining with CD138-APC (BD, 281–2), followed by magnetic enrichment using anti-APC MicroBeads (Miltenyi # 130-090-855). For RNA-seq, GL7+ cells were further enriched from the CD138-depleted fractions using GL7-PE (Biolegend #144608) and anti-PE MicroBeads (Miltenyi #130-105-639).
Flow cytometry was performed on a BD LSRII using FACSDiva (v6.2) and analyzed using FlowJo software. The following gating strategy preceded all flow cytometry analyses presented. Cells were gated on 1) lymphocytes (forward light scatter [FSC]–area by side scatter [SSC]–area), 2) singlets (FSC-height by FSC- width), 3) singlets (SSC- height by SSC-width), 4) live cells (Viability Dye negative), with 5) the exclusion of contaminating macrophages bearing CD11b (Supplemental Figure2A).

Liang F.T, & Philipp M.T. (2000). Epitope Mapping of the Immunodominant Invariable Region of Borrelia burgdorferi VlsE in Three Host Species. Infection and Immunity, 68(4), 2349-2352.

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