Cd5 bv421

Manufactured by BioLegend

CD5-BV421 is a fluorescently-labeled antibody that binds to the CD5 cell surface antigen. CD5 is expressed on T cells and a subset of B cells. The BV421 fluorescent label allows for detection and analysis of CD5-expressing cells using flow cytometry.

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3 protocols using cd5 bv421

Immune Cell Characterization by Flow Cytometry

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The procedure used in this study has been published previously (14 (link),26 (link)). Briefly, the following anti-mouse monoclonal antibodies were purchased from BD Biosciences: Ly6G-peridinin chlorophyll protein (PerCP)-Cy5.5 (1A8; 560602), Ly6C-fluorescein isothiocyanate (FITC) (AL-21; 553104), CD4-allophycocyanin (APC) (RM4-5; 553051), CD25-FITC (7D4; 553071), and CD19-APC (1D3; 550992). The following antibodies were purchased from BioLegend: CD45-FITC (30F-11;103108), CD45-PerCP-Cy5.5 (30F-11;103132), CD45-brilliant violet (BV510) (30F-11;103138), CD11b-APC/Fire750 (M1/70;101262), CD244-Alexa Fluor 647 (2B4;133509), F4/80-phycoerythrin (PE) (BM8;123110), PD-L1-BV421 (10F.9G2;124315), CD8a-APC/Fire 750 (53-6.7;100766), CD8a-PE (53-6.7; 100708), CD127-BV510 (A7R34;135033), CD1d-PE (1B1;123509), and CD5-BV421 (53-7.3;100618). The isolated immune cells were stained in a 96-round-bottom-well plate for 20 min at 4°C and washed with PBS containing 1% bovine serum albumin. Sorting of Ly6G⁺CD244⁺ and Ly6G⁺CD244⁻ cells was conducted on a FACSAria instrument (BD Biosciences). Flow cytometric data were obtained by a FACS Verse instrument (BD Biosciences) and analyzed by FlowJo software (TreeStar, Inc.). CD45-gated cells were analyzed in this study.

Sugita Y., Yamashita K., Fujita M., Saito M., Yamada K., Agawa K., Watanabe A., Fukuoka E., Hasegawa H., Kanaji S., Oshikiri T., Matsuda T., Nakamura T., Suzuki S, & Kakeji Y. (2021). CD244+ polymorphonuclear myeloid-derived suppressor cells reflect the status of peritoneal dissemination in a colon cancer mouse model. Oncology Reports, 45(6), 106.

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Quantifying Lung Inflammatory Cells

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To assess and quantify inflammatory cells infiltration, lungs of ADAM28^+/+ and ADAM28^-/- were harvested and digested in collagenase C (1mg/ml; Gibco) prior to red blood cell lysis (Red Blood Cell Lysis Buffer, Sigma Aldrich, Saint-Louis, Missouri). Cells were stained with fluorochrome-conjugated surface antibodies during 30 minutes, fixed and permeabilized using Cytofix/Cytoperm (ref#554714 BD Biosciences, Erembodegem, Belgium) before intracellular antigen staining. Antibodies used for flow cytometry analysis were: CD5-BV421 (ref#53-7.3, BioLegend), CD4-PERCP CY 5.5 (ref#RM4-5, BD Biosciences), CD8a-BB515 (ref#53-6.7, BD Biosciences), IFN-γ-PE CY7 (ref#XMG 1.2, BD Biosciences), IL-4-APC (ref#11B11, eBioscience, Thermo Fisher Scientfic), CD45R-APC-eFluor780 (B220) (ref#RA3-6B2, eBioscience). Data were acquired on FACS CANTO II flow cytometer (BD Biosciences) and analyzed using BD FACSDiva software (BD Biosciences).

Bendavid G., Hubeau C., Perin F., Gillard A., Nokin M.J., Carnet O., Gerard C., Noel A., Lefebvre P., Rocks N, & Cataldo D. (2023). Role for the metalloproteinase ADAM28 in the control of airway inflammation, remodelling and responsiveness in asthma. Frontiers in Immunology, 13, 1067779.

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Multiparametric Flow Cytometry Analysis

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Following collection of patient specimens, the following subsets were analyzed by FCM: whole BM, BMNCs, CD138+ cells, and CD138− cells. For each sample, at least 200,000 cells were washed and incubated for 30 min at 4°C with CD138-PeCy7, CD38-BV421, CD45-APC, CD19-PE, and CD56-FITC, or matching isotype controls (BioLegend), at a 1:200 dilution. Stained cells were washed once and fixed with 1% paraformaldehyde (PFA). For analysis of previously frozen primary CD138+ and CD138− cells, BMNCs, T cells, and B cells, Human TruStain FcX Fc Receptor Blocking Solution (BioLegend) was used according to the manufacturer’s directions. Cells were then stained with CD5-BV421, CD147-PE/Cy7, CD166-PE, CD205-APC (BioLegend), and CD98hc-FITC (ThermoFisher), or matching isotype controls (BioLegend). All samples were analyzed on a BD LSRFortessa X-20 flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (FlowJo LLC).

Oldham R.A., Faber M.L., Keppel T.R., Buchberger A.R., Waas M., Hari P., Gundry R.L, & Medin J.A. (2020). Discovery and validation of surface N-glycoproteins in MM cell lines and patient samples uncovers immunotherapy targets. Journal for Immunotherapy of Cancer, 8(2), e000915.

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