Cd19 v450

Manufactured by BD

Sourced in United Kingdom

The CD19-V450 is a flow cytometry reagent that binds to the CD19 antigen expressed on B cells. It is used for the identification and enumeration of CD19-positive cells in biological samples.

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Anti-CD19-V450 (4 citations) V450-coupled anti-CD19 (2 citations) V450 conjugated anti-CD19 clone SJ25C1 (2 citations) Anti-human CD19-V450 HIB19 (2 citations)

13 protocols using cd19 v450

Comprehensive Immune Cell Profiling

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The following antibodies were used: CD4-V500, CD11c-PE-Cy7, CD19-V450, CD19-APC-Cy7, CD69-PE-Cy7, IgM-PerCP-Cy5.5, CD45.2-V500, CD45.2-APC (BD Biosciences), IgD-PE, CD45.1-FITC, CD11b-PerCP-Cy5.5, CD21-PB, CD23-PE-Cy7, AA4.1-APC (eBiosciences), and CD86-PB, CD45.1-PB (BioLegend). Following red blood cell lysis, Fc receptor blockade, and staining, cells were processed on a BD FACSVerse or LSR II flow cytometer and analyzed using FlowJo v9.6.4 (Treestar).

Mills R.E., Lam V.C., Tan A., Cresalia N., Oksenberg N., Zikherman J., Anderson M., Weiss A, & Hermiston M.L. (2015). Unbiased modifier screen reveals that signal strength determines the regulatory role murine TLR9 plays in autoantibody production. Journal of immunology (Baltimore, Md. : 1950), 194(8), 3675-3686.

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Multiparameter Flow Cytometry Analysis of Bone Marrow Plasma Cells

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Fresh BM samples were stained with the following mouse anti-human monoclonal antibodies according to the manufacturer`s recommendations: CD38-FITC or APC, CD56-PE, CD27-peridinin chlorophyll protein complex with cyanin-5.5 (PerCP-Cy5.5), CD138-PerCP-Cy5.5 or APC, CD117- phycoerythrin-cyanin-7 (Pe-Cy7), CD81-allophycocyanin-Hilite® 7 (APC-H7), CD19-V450, CD45-V450, cytIgLambda-FITC, cytIgKappa-PE-Cy7 (all MAbs were obtained from BD Biosciences). For intracellular staining, samples were incubated with commercially available BD FACS Permeabilizing Solution 2 (BD Biosciences) according to manufacturer`s instructions. Samples were analyzed on FACSCanto II flow cytometer (Becton Dickinson) using FACSDiva software, respectively (Becton Dickinson). The instrument was daily calibrated with calibration beads (Cytometer Setup and Tracking from BD Biosciences). At least 10⁶ gated BM cells per sample were collected for the assay to attain the sensitivity 0.01% (i.e. 10^-4). Myeloma PCs were indicated as CD45^dimCD38⁺CD138⁺CD56⁺CD19^—CD117⁺CD27^—CD81^— phenotype gate and were presented as the percentage of all nucleated BM cells. Gating strategy is presented in Supplementary Figure 1.

Batorov E.V., Tikhonova M.A., Pronkina N.V., Kryuchkova I.V., Sergeevicheva V.V., Sizikova S.A., Ushakova G.Y., Aristova T.A., Batorova D.S., Menyaeva E.V., Gilevich A.V., Shevela E.Y., Ostanin A.A, & Chernykh E.R. (2018). Increased circulating CD4+FOXP3+ T cells associate with early relapse following autologous hematopoietic stem cell transplantation in multiple myeloma patients. Oncotarget, 9(43), 27305-27317.

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Immunophenotyping of Hairy Cell Leukemia

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HCL samples were stained for CD19-eFluor450 (eBioscience, Hatfield, UK)/CD19-V450 (BD), CD11c-APC (BioLegend, London, UK), CD103-FITC (BioLegend/BD), CD27-APC-eFluor780 (eBioscience) and CD3-PE (BD/eBioscience) as described for immunophenotyping. HCLc tumour cells were sorted and selected as a CD19⁺CD11c^HICD103⁺CD27^- population and germline T cells from the same sample as CD3⁺ using the FACS Aria I (BD) (Fig 1). Sorted populations were re-analysed for purity on the FACS Aria I: purity of HCLc sorts was 92.9–98.3% and of T cells 96.7–100% (Fig 1 is a typical example). Data were analysed using FACS Diva (BD) and FlowJo (TreeStar).

Weston-Bell N.J., Tapper W., Gibson J., Bryant D., Moreno Y., John M., Ennis S., Kluin-Nelemans H.C., Collins A.R, & Sahota S.S. (2016). Exome Sequencing in Classic Hairy Cell Leukaemia Reveals Widespread Variation in Acquired Somatic Mutations between Individual Tumours Apart from the Signature BRAF V(600)E Lesion. PLoS ONE, 11(2), e0149162.

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Whole Blood Leukocyte Immunophenotyping

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For this study, 50 μL whole blood samples were stained with leukocyte marker antibodies using an established 5-fluorophore-6-marker surface staining method, as previously described [14 (link)]. In brief, whole blood was incubated with a cocktail of monoclonal mouse anti-human antibodies: CD2-APC, CD36-PE, CD16-APC-H7, CD45-AmCyan, CD19-V450 and rat anti-human chemoattractant receptor-homologous molecule expressed on T helper type 2 (Th2) cells (CRTh2)-AF647 (all BD Biosciences). Red blood cells were lysed with BD FACS Lysing Solution before fixation in Stabilizing Fixative and transfer to TruCOUNT Tubes (all BD Biosciences).

Hibbert J., Strunk T., Nathan E., Prosser A., Doherty D., Simmer K., Richmond P., Burgner D, & Currie A. (2022). Composition of early life leukocyte populations in preterm infants with and without late-onset sepsis. PLoS ONE, 17(3), e0264768.

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Immunophenotyping of Mononuclear Cells

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Patient sample mononuclear cells that had undergone Ficoll gradient isolation were immunophenotyped to standard clinical specifications by the OHSU Histopathology Shared Resource laboratory. The following antibodies were used: CD3-FITC (#349201; BD Biosciences, Franklin Lakes, NJ), CD5-PC-Cy7 (#348790; BD Biosciences), CD14-APC-H7 (#643077; BD Biosciences), CD19-V450 (#644492; BD Biosciences), CD33-PerCP-Cy-5.5 (#341640; BD Biosciences), CD45-Pacific-Orange (#MHCD4530; Invitrogen, Carlsbad, CA), CD64-PE (#558592; BD Biosciences), and CSF1R-APC (#347306; BioLegend, San Diego, CA). Surface marker analysis was performed on a BD FACSCanto II flow cytometer and the data were analyzed using FlowJo (FlowJo, LLC, Ashland, OR).

Edwards V D.K., Sweeney D.T., Ho H., Eide C.A., Rofelty A., Agarwal A., Liu S.Q., Danilov A.V., Lee P., Chantry D., McWeeney S.K., Druker B.J., Tyner J.W., Spurgeon S.E, & Loriaux M.M. (2018). Targeting of colony-stimulating factor 1 receptor (CSF1R) in the CLL microenvironment yields antineoplastic activity in primary patient samples. Oncotarget, 9(37), 24576-24589.

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Immunomodulatory Effects of LILRA3

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For microarray analysis, 10⁶/mL PBMCs were cultured for 24 h in the absence or presence of 1 µg/mL baculovirus LILRA3 in a 25 cm² suspension culture flask. The cells were pelleted and their total RNA isolated using the Qiagen RNeasy Minikit according to the manufacturer’s instructions. Microarray and data processing are detailed below and deposited in Gene Expression Omnibus (accession GSE61356). To confirm the microarray data, PBMCs were stimulated overnight with 100, 500 and 1000 ng/mL of mammalian LILRA3 and analyzed for IL-6, IL-1A, IL-1B, IL-10 and LILRA3 expression. For changes in B-cell and monocyte expression levels of MHC and costimulatory monecules, PBMCs incubated for 48 h with 0–1000 ng/mL baculovirus LILRA3 were stained with CD14-APC-CY7 (Biolegend), CD19-V450, CD3-V500, HLA-ABC-FITC or CD80-FITC, CD86-PE (all BD Bioscience) or HLA-DR-PE (Beckman Coulter), and analyzed via flow cytometry.

Low H.Z., Ahrenstorf G., Pommerenke C., Habermann N., Schughart K., Ordóñez D., Stripecke R., Wilk E, & Witte T. (2016). TLR8 regulation of LILRA3 in monocytes is abrogated in human immunodeficiency virus infection and correlates to CD4 counts and virus loads. Retrovirology, 13, 15.

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Multiparameter Flow Cytometric Immune Profiling

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For flow cytometric characterisation of leukocytes from spleen, blood, BM, lung tissue, and BALF the following antibodies were used: F4/80 FITC (1:100, AB_893500, clone BM8; BioLegend), CD3ε Pacific Blue (1:100, AB_397063, clone 500A2), CD3ε PerCPCy5.5 (1:100, AB_10562558, clone 145-2C11), CD11b PE-Cy7 (1:400, AB_394491, clone M1/70), CD19 V450 (1:100, AB_1645269, clone 1D3), Ly6G FITC (1:100, AB_10562567, clone 1A8), Siglec-F PE (1:200, AB_394341, clone E50-2440; all BD Bioscience), Ly6G APC (1:1600, 17-9668-82, clone 1A8; eBioscience). Flow cytometry was performed with the FACS Canto II (BD Bioscience) and the obtained data were analysed using FlowJo 7.6.1 (FlowJo).

Mothes B., Bucher K., Ammon-Treiber S., Schwab M., Piekorz R.P., Hirsch E., Nürnberg B, & Beer-Hammer S. (2016). p110γ/δ Double-Deficiency Induces Eosinophilia and IgE Production but Protects from OVA-Induced Airway Inflammation. PLoS ONE, 11(7), e0159310.

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Immunophenotyping of Murine Splenocytes

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Cells used for immunophenotyping studies were either splenocytes or leukocytes obtained from blood. When mice were euthanized, blood was collected via cardiac puncture using a syringe that had been rinsed with 10% potassium ethylenediaminetetraacetic acid. The blood was placed in a 1 mL Wintrobe tube (Fisher Scientific, Hampton, New Hampshire) and centrifuged at 2000 rpm for 10 minutes. The plasma was removed and stored at −20°C for future analysis. The buffy coat was collected and placed in 2 mL of red blood cell solution for 10 minutes at room temperature. Then 10 mL of PBS was added and the tubes were centrifuged at 1500 rpm for 5 minutes. The cells present in the pellet or splenocytes were incubated with a panel of labeled monoclonal antibodies: anti-CD3 PE-Cy7, CD4 BV605, CD8 APC-H7, natural killer (NK)1.1 APC, and CD19 V450 (BD Biosciences, San Jose, California) in staining buffer on ice in the dark for 20 minutes. Cells were then washed in the staining buffer and resuspended in staining buffer and analyzed on an LSRII flow cytometer (BD Biosciences). Isotype controls were used for each experiment. Results were analyzed using FlowJo version 10 software (FlowJo, LLC, Ashland, Oregon).

Beseme S., Fast L., Bengston W., Turner M., Radin D, & McMichael J. (2020). Effects Induced In Vivo by Exposure to Magnetic Signals Derived From a Healing Technique. Dose-Response, 18(1), 1559325820907741.

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Leukocyte Profiling from Human Blood

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Human blood samples (0.5–3mL) were transferred into 0.1 M EDTA coated 50 mL lab tubes. Leucocytes fraction was obtained after incubating the blood samples for 2.5 minutes in room temperature with BD FACS Lysing Solution (BD bioscience, San Jose, California). Leucocytes were stained with FACS buffer containing the following conjugated antibodies (10 minutes on ice in the dark): CD3-V450, CD19-V450, CD56-V450, HLA-DR1-APC, CD14-APC-Cy7 and CD16-FITC (BD Bioscience). Stained samples were further washed and filtered using 70μm mesh before analyzed by FACS (FACS Canto II; BD Biosciences, San Jose, California) and the FlowJo software.

Pecht T., Haim Y., Bashan N., Shapiro H., Harman-Boehm I., Kirshtein B., Clément K., Shai I, & Rudich A. (2016). Circulating Blood Monocyte Subclasses and Lipid-Laden Adipose Tissue Macrophages in Human Obesity. PLoS ONE, 11(7), e0159350.

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Characterizing Lymph Node Immune Cells

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To analyze immune cell populations in the draining lymph nodes, the cells were isolated by slitting the organs and pressing them through 100 µm mesh cups (Corning, # 52360) to generate single-cell suspensions. 2 × 10⁶ cells were stained with different combination of the following antibodies: CD11c-FITC (# 553801), CD4-PE (# 553730), CD27-PE (# 558754), NK1.1-PE-Cy7 (# 552878), 7AAD (# 559925) CD19-V450, (# 560375, CD11b-V450 (# 560455) all from BD Biosciences; CD3e-APC (# 17–0031) from eBioscience ; AnnexinV-FITC from Immunotools (# 31490013) and CD8a-FITC from Miltenyi (# 130–102–806). A minimum of 5 × 10⁵ events were detected per measurement. Flow cytometry was performed on a FACS Canto II (BD Biosciences, Heidelberg, Germany) and analyzed using FlowJo Software v7.6.5 (Treestar). The gating strategy is representatively depicted in Fig. S2.

Finkel P., Frey B., Mayer F., Bösl K., Werthmöller N., Mackensen A., Gaipl U.S, & Ullrich E. (2016). The dual role of NK cells in antitumor reactions triggered by ionizing radiation in combination with hyperthermia. Oncoimmunology, 5(6), e1101206.

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