Cd5 53 7

Manufactured by BioLegend

CD5 (53-7.3) is a mouse monoclonal antibody that recognizes the CD5 antigen. CD5 is a transmembrane glycoprotein that is expressed on the surface of T cells, a subset of B cells, and some thymocytes. It functions as a co-receptor during T cell activation.

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3 protocols using cd5 53 7

Multiparametric Flow Cytometry Analysis

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Cell suspensions from mouse liver, BM (one femur and tibia), spleen, thymus, mesenteric lymph nodes, and peritoneal cavity were processed as previously described (47 (link)), and data were acquired on a FACSCanto10c (BD) and analyzed with FlowJo Software (Tree Star).
The following anti-mouse antibodies were used: B220 (RA3-6B2, BioLegend), BAFFR (7H22-E16,BD), BP-1 (6C3, BioLegend), CD3e (145-2C11, eBioscience), CD4 (GK1.5, eBioscience), CD5 (53-7.3, BioLegend), CD8 (53-6.7, BioLegend), CD9 (MZ3, BioLegend), CD11b (M1/70, eBioscience), CD19 (6D5, 1D3, BioLegend), CD21/CD35 (7E9, BioLegend), CD23 (B3B4, BioLegend), CD24 (M1/69, BioLegend), CD25 (PC61, Biolgend), CD43 (S7, BioLegend), CD44 (IM7, BioLegend), CD45.1 (A20, BioLegend), CD45.2 (104, BioLegend), CD69 (H1.2F3, BioLegend), CD80 (16-10A1, BioLegend), CD86 (GL1, BioLegend), CD93 (AA4.1, BioLegend), DAPI (BIOTIUM), Gr-1 (RB6-8C5, eBioscience), IgDa (AMS-9.1, BioLegend), IgD (11-26c, BioLegend), IgMa (DS-1, MA-69, BioLegend), IgM (Il/41, RMM-1, BioLegend), TCRγδ (GL3, BioLegend), Live dead dye (Zombie Aqua Dye, BioLegend), Live dead dye (Zombie NIR, BioLegend), pERK (4B11B69, BD), pPLCγ2 (K86-1161, BD), Lin28b (AP1485C, ABGENT), and anti-rabbit IgG (BioLegend).

Xu X., Deobagkar-Lele M., Bull K.R., Crockford T.L., Mead A.J., Cribbs A.P., Sims D., Anzilotti C, & Cornall R.J. (2020). An ontogenetic switch drives the positive and negative selection of B cells. Proceedings of the National Academy of Sciences of the United States of America, 117(7), 3718-3727.

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Multicolor Immunofluorescence of Murine Tissues

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Murine tissue samples were fixed with 1% (w/v) paraformaldehyde (Electron Microscopy Services) in PBS for 16 h, washed with PBS, and equilibrated in 30% (w/v) sucrose for a further 16 h. Tissues were then frozen at −80 °C in Optimal Cutting Temperature (OCT) embedding medium (Thermo Fisher Scientific). 20um cryostat sections air-dried for 30 min and then permeabilised and blocked with blocking buffer (0.1 M TRIS), containing 0.1% Triton X-100 (Sigma), 1% (w/v) normal rat serum, 1% (w/v) BSA (R&D Systems, Abingdon, UK) for 1 hour at RT. Sections were stained overnight at 4 °C with a combination of the following antibodies in blocking buffer: CD19 (6D5; Biolegend, dilution: 1:25, RRID: AB_11218994, Cat. No. 115543), CD31 (MEC13.3; Biolegend, dilution: 1:50, RRID: AB_2563319, Cat. No. 100237), CD5 (53-7.3; Biolegend, dilution: 1:25, RRID: AB_2563930, Cat. No. 100627).
Slides were mounted using Vectashield Mounting Medium without or with DAPI (Vector Laboratories Inc., USA). Images were acquired using TCS SP8 (Leica, Milton Keynes, UK) confocal microscope with ZEN 3.7 software (Zeiss, Cambourne, UK). Raw imaging data were processed and quantified using Imaris v9.3 (Bitplane, Zurich, Switzerland).

Suchanek O., Ferdinand J.R., Tuong Z.K., Wijeyesinghe S., Chandra A., Clauder A.K., Almeida L.N., Clare S., Harcourt K., Ward C.J., Bashford-Rogers R., Lawley T., Manz R.A., Okkenhaug K., Masopust D, & Clatworthy M.R. (2023). Tissue-resident B cells orchestrate macrophage polarisation and function. Nature Communications, 14, 7081.

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Comprehensive Cellular Flow Cytometry Protocol

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For cellular flow cytometry, mAbs included the following: Thy1.2/CD90 (30-H12, BioLegend), TCRβ (H57, eBioscience), Vα2 (B20.1, BD), CD3ε (145-2C11, BioLegend), CD8α (53.6.7, BioLegend), CD8β (53-5.8, BioLegend), CD4 (GK1.5, BD Biosciences), CD69 (H1.2F3, BioLegend), CD24 (M1/69, BioLegend), and CD5 (53-7.3, BioLegend). Other binding reagents included the following: peanut agglutinin (PNA)-FITC (Sigma-Aldrich), streptavidin-PE and streptavidin-APC-Cy7 (BioLegend), H-2Kb-SIINFEKL (OVA) tetramer, and thymic leukemia antigen (TLA) tetramers (NIH tetramer core facility). For multiplex IP and PiSCES analysis, antimouse Abs are listed in table S1, whereas a similar table for antihuman Abs used in conjunction with Jurkat and JRT3 cell lines was previously published (40 (link)).

Neier S.C., Ferrer A., Wilton K.M., Smith S.E., Kelcher A.M., Pavelko K.D., Canfield J.M., Davis T.R., Stiles R.J., Chen Z., McCluskey J., Burrows S.R., Rossjohn J., Hebrink D.M., Carmona E.M., Limper A.H., Kappes D.J., Wettstein P.J., Johnson A.J., Pease L.R., Daniels M.A., Neuhauser C., Gil D, & Schrum A.G. (2019). The early proximal αβ TCR signalosome specifies thymic selection outcome through a quantitative protein interaction network. Science immunology, 4(32), eaal2201.

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