Ly6c bv650

Manufactured by BioLegend

Sourced in Belgium

Ly6C-BV650 is a fluorochrome-conjugated antibody that binds to the Ly6C antigen, which is expressed on certain immune cell populations. This product can be used for the identification and analysis of Ly6C-positive cells in flow cytometry applications.

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3 protocols using ly6c bv650

Flow Cytometric Identification of Hepatic Immune Cells

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Cells were prestained with a 1:100 dilution of Zombie Aqua (Fixable Viability Dye; BioLegend, London, UK) for 20 minutes at 4°C in the dark. After 10 minutes, an equal volume of a 1:100 dilution of TruStain FcX PLUS (anti-mouse CD16/32 antibody) and True-Stain Monocyte Blocker (BioLegend) was added. After a washing step, cells were stained with CD3e-PerCP-Cy5.5, CD19-PerCP-Cy5.5 (eBioscience, Thermo Fisher Scientific), NK1.1-PerCP-Cy5.5, CD103-PerCP-Cy5.5, F4/80-FITC, Ly6G-BV785, Ly6C-BV650 (BioLegend), SiglecF-PerCP-Cy5.5, CD45-APC-Cy7, CD11b-PE-Cy7 and Tim4-PE (BD Biosciences, Erembodegem, Belgium) for 20 minutes at 4°C in the dark. Cells were analyzed with a BD FACSAria Fusion flow cytometer (BD Biosciences) and FlowJo software (FlowJo LLC, BD Biosciences), and gated first as live CD45⁺ single cells. Subsequently, CD3e⁺, CD19⁺, NK1.1⁺, CD103⁺ and SiglecF⁺ cells were eliminated, and CD11b⁺Ly6C⁺Ly6G^- monocytes, CD11b⁺Ly6C^-F4/80⁺Tim4⁺ Kupffer cells (KCs) and CD11b⁺Ly6C^-F4/80⁺Tim4^- monocyte-derived macrophages (MoMfs) were gated.

Vanderborght B., De Muynck K., Lefere S., Geerts A., Degroote H., Verhelst X., Van Vlierberghe H, & Devisscher L. (2020). Effect of isoform-specific HIF-1α and HIF-2α antisense oligonucleotides on tumorigenesis, inflammation and fibrosis in a hepatocellular carcinoma mouse model. Oncotarget, 11(48), 4504-4520.

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Phenotyping Fungal Lung Infection

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C57BL/6 mice (Jackson) were infected with 10⁶ Uvitex 2B-labeled (10 µg/ml; 5 min) yeast cells administered intratracheally. Single cell suspensions were made from harvested lungs with a 70-µm cell strainer and treatment with collagenase D (1 mg/ml; Roche) and DNase (50 U/ml; Roche). Leukocytes were enriched by density sedimentation (60%/40% Percoll, 20 min, relative centrifugal force of 600) and collection of cells at the interface. Approximately 10⁶ cells were stained for cellular markers and fixed with 4% paraformaldehyde (30 min). The markers included CD64-fluorescein isothiocyanate, CD45-peridinin chlorophyll protein-Cy5.5, SiglecF-phycoerythrin-Cy7, Ly6C-BV650, CD11c-BV786, CD90.2-allophycocyanin (APC), B220-APC, MHCII-A700, Ly6G-BUV395 (all from BioLegend), and Near IR live/dead stain (Thermo, Fisher). Cells were analyzed with an LSRII flow cytometer (BD Biosciences), and data were processed with FlowJo software (version 10.1).

Garfoot A.L., Goughenour K.D., Wüthrich M., Rajaram M.V., Schlesinger L.S., Klein B.S, & Rappleye C.A. (2018). O-Mannosylation of Proteins Enables Histoplasma Yeast Survival at Mammalian Body Temperatures. mBio, 9(1), e02121-17.

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Multiparametric Spleen Cell Analysis

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Mouse spleens were first disrupted in R10 (RPMI with 10% FBS) containing 1mg/ml collagenase D (Roche collagenase D). After 30 min, the suspension was passed through a 70 μm cell strainer and then lysed with the ACK lysis buffer system to remove erythrocytes (Lonza). The splenocytes were then washed in PBS and then stained with viability dye (Aqua or Blue Viability at 0.025 mg/ml; Thermofisher) before our staining panels were applied. Staining panels were derived from the following fluorescently conjugated anti-murine antibodies, each used at a final dilution of 1:100 in PBS: CD11c-PE Cy7 (N418, Biolegend); B220-BV605 (RA3–6B2, BD Horizon); B220-FITC (RA3–6B2, BD PharMingen); MHCII-BV510 (M5/114.15.2, Biolegend); CD3-BV785 (17A2, Biolegend); CD19-PerCP/Cy5.5 (6D5, Biolegend); IL6R-APC (D7715A7, Biolegend); CD11b-APC (M1/70, Biolegend); B220-FITC (RA3–6B2, BD PharMingen); CD138-BV421 (281–2, BD Horizon); gp130-APC (KGP130, eBioscience); CD11b-FITC (M1/70, Biolegend); Siglec-H-APC (551, Biolegend); CD93 (AA4.1, Biolegend); CD8α-BV605 (53–6.7, Biolegend); DCIR2-PE (33D1, Biolegend); Ly6C-BV650 (HK1.4, Biolegend); F4/80-Alexa 488 (BM8, Thermofisher). Samples were assayed on a 5 laser LSR Fortessa (BD Biosciences) and data was analyzed using FlowJo software version 9.3.2 (TreeStar).

Yousif A.S., Ronsard L., Shah P., Omatsu T., Sangesland M., Bracamonte Moreno T., Lam E.C., Vrbanac V.D., Balazs A.B., Reinecker H.C, & Lingwood D. (2020). The persistence of interleukin-6 is regulated by a blood buffer system derived from dendritic cells. Immunity, 54(2), 235-246.e5.

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