Cd4 buv395

Manufactured by BD

Sourced in United States

The CD4-BUV395 is a fluorochrome-conjugated antibody that binds to the CD4 cell surface antigen. It is designed for use in flow cytometry applications to identify and quantify CD4-positive cells in biological samples.

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Lab products found in correlation

Cd3 fitc, by BD (9 mentions) Live dead ghost dye 780, by Cytek Biosciences (7 mentions) Cd8 buv805, by BD (7 mentions) Pd1 pecf594, by BD (6 mentions) Cd3 buv737, by BD (5 mentions) 41bb percpef710, by Thermo Fisher Scientific (5 mentions) Lag3 bv711, by BD (4 mentions) Tim3 apc, by Thermo Fisher Scientific (4 mentions) Cd8 percp cy5, by BioLegend (4 mentions) Cd8 buv395, by BD (3 mentions) Cd8 bv786, by BD (3 mentions) Lsrfortessa flow cytometer, by BD (3 mentions) Cd8 bv421, by BioLegend (2 mentions) Anti human cd3 buv737, by BD (2 mentions) Gitr bv605, by BD (2 mentions) Ctla 4 bv786, by BioLegend (2 mentions) Cd3 bv786, by BD (2 mentions) Hla dr af700, by BioLegend (2 mentions) Cd45 v450, by BD (2 mentions) Cd19 buv737, by BD (2 mentions)

Close spelling variants of this product

Anti-CD4-BUV395 (14 citations) CD4-BUV395 (clone SK3), (4 citations) BUV395-conjugated anti-mouse CD4 (2 citations) Anti-CD4-BUV395 (clone GK1.5, (2 citations)

36 protocols using cd4 buv395

Multiparametric Flow Cytometry Profiling of PBMCs

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For surface staining, PBMCs were incubated with directly conjugated antibodies for 30 min at 4°C. Antibodies used included anti-human CD3-BUV737 (clone VCHT1), CD3-BV786 (clone SK7), CD4-BUV395 (clone RPA-T4), CD8-BUV395 (clone RPA-T8), GITR-BV605 (clone V27-580), CD38-BUV737 (clone HB7), CD25-PE-CF594 (clone M-A251, BD Biosciences, San Diego, CA, USA), CD4-APC-fire750 (clone SK3), CD8-BV421 (clone RPA-T8), αβTCR-BV421 (clone IP26), CD56-AF700 (clone 5.1H11), CD56-APC (clone 5.1H11), FasL-PE-Cy7 (clone NDK-1), CTLA-4-BV786 (clone BNI3), CD73-BV711 (clone AD2), HLA-DR-AF700 (clone L243, BioLegend, San Diego, CA, USA), LAG3-APC (clone 3DS223H, Invitrogen, Carlsbad, CA, USA) and the corresponding isotype controls. For intracellular staining of Foxp3 (clone 25901C7, BD Biosciences), Granzyme A-PE-cy7 (clone CB9), granzyme B-APC-fire750 (clone A16A02), perforin-PE-CF594 (clone dG9), Ki-67-BV711 (clone Ki-67, BioLegend) and IDO-APC (clone eyedio, Invitrogen) cells were fixed and permeabilized using Foxp3 Staining Buffer Set (BD Biosciences) according to the manufacturer’s recommendations. A fixable viability dye eFluor 506 (Ebioscience, San Diego, CA, USA) was used to assess cell viability. Data were acquired on a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Zhang L., Wei Y., Wang D., Du J., Wang X., Li B., Jiang M., Zhang M., Chen N., Deng M., Song C., Chen D., Wu L., Xiao J., Liang H., Zhao H, & Kong Y. (2022). Elevated Foxp3+ double-negative T cells are associated with disease progression during HIV infection. Frontiers in Immunology, 13, 947647.

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Immunophenotyping of AML Leukapheresis Samples

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Mononuclear cells from the peripheral blood of 36 patients with AML indicated at leukapheresis due to hyperleukocytosis at diagnosis were obtained by separation of leukapheretic products on Histopaque 1077 (Sigma, Prague, Czech Republic). The cells were resuspended in RPMI 1640 medium with 10% fetal calf serum and with antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin), and aliquots were used for RNA isolation and for analysis of surface markers by flow cytometry. Additional analyses of PD-L1 transcripts were performed from cDNA samples, which had been taken for routine clinical analyses and cryopreserved. A written informed consent with the use of biological material for research purposes was obtained from all patients. The study was approved by the Ethics Committee of the Institute of Hematology and Blood Transfusion of the Czech Republic as a part of the research project 16-30268A (June 2015).
Antibodies used were the following: CD45-V450 (#560367), CD4-BUV395 (#564724), CD8-BUV395 (#563795), CD19-BUV737 (#564303), CD123-BUV395 (#564195), CD371(CLL-1)-BB515 (#565926), CD135(FLT3)-AlexaFluor647 (#563494), and CD34-BV786 (#743534) were from BD Biosciences (Prague, Czech Republic); CD38-PE (#1P-366-T100) from Exbio (Prague, Czech Republic); PD-1-APC (#17-2799-42) and PD-L1-PE-Cy7 (#25-5983-42) from Affymetrix, Inc. (San Diego, CA, USA).

Brodská B., Otevřelová P., Šálek C., Fuchs O., Gašová Z, & Kuželová K. (2019). High PD-L1 Expression Predicts for Worse Outcome of Leukemia Patients with Concomitant NPM1 and FLT3 Mutations. International Journal of Molecular Sciences, 20(11), 2823.

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Multiparameter Flow Cytometry Phenotyping

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Standard extracellular staining (20 mins at 24°C) was performed using the following fluorophore-labeled antibodies: CD3-BUV737 (BD Biosciences), CD4-BUV395 (BD Biosciences), CD8-BV711 (BD Biosciences), CD14-BV510 (BD Biosciences), CD19-BV510 (BioLegend), Live/Dead Fixable Aqua (ThermoFisher), CCR7-PE-CF594 (BD Biosciences), CD45RA-APC-H7 (BD Biosciences), CD28-APC-R700 (BD Biosciences), CD57-PE (BioLegend), PD-1-BV605, BV421 (BioLegend), CD69-BV650 (BioLegend), CD38-BV421 (BioLegend), HLA-DR-PerCP-Cy5.5 (BD Biosciences), TIGIT-PE-Cy7 (BioLegend), DNAM-BB515 (BD Biosciences), FcγRIIB-BV786 (BD Biosciences). Fluorescence minus one (FMO) controls were used to determine negative expression of FcγRIIB. Apoptosis was measured with Caspase-3/7 Green Flow Cytometry Assay Kit (ThermoFisher) or FITC Annexin V with 7-AAD Viability Staining Solution (BioLegend), according to the manufacturer’s instructions.

Sun H., Hartigan C.R., Chen C.W., Sun Y., Tariq M., Robertson J.M., Krummey S.M., Mehta A.K, & Ford M.L. (2021). TIGIT regulates apoptosis of risky memory T cell subsets implicated in belatacept-resistant rejection. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 21(10), 3256-3267.

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Multiparametric Flow Cytometry Protocol

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Single-cell suspensions were incubated in anti-CD16/CD32 Fc block antibody (5 μg/ml; clone 2.4G2; cat # 553141, BD Biosciences, San Jose, CA) and fixable viability dye eFluor 780 (Invitrogen, Carlsbad, CA) for 15 min at 4 °C in cold PBS. Cells were then washed once and stained with combinations of the following antibodies: CD45-BV510 (2 μg/ml; clone 30-F11; cat # 103138, BioLegend, San Diego, CA), TCRβ-PerCP-Cy5.5 (2 μg/ml; clone H57-597; cat # 109228, BioLegend, San Diego, CA), CD4-BUV395 (2 μg/ml; clone GK1.5; cat # 563790, BD Biosciences, San Jose, CA), CD8-af700 (5 μg/ml; clone KT15; cat # MCA609A700, Bio-Rad, Hercules, CA), NKp46-APC (1 μg/ml; clone 29A1.4; cat # 137607, BioLegend, San Diego, CA), CD49b-PE (2 μg/ml; clone DX5; cat # 108908, BioLegend, San Diego, CA), B220-BUV661 (2 μg/ml; clone RA3-6B2; cat # 612972, BD Biosciences, San Jose, CA), and Thy1.2-BUV805 (1 μg/ml; clone 53-2.1; cat # 741908, BD Biosciences, San Jose, CA). Data were acquired on a Symphony flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR). Gating strategies for the identification of CD8⁺ T cells and NK cells are provided in Supplementary Fig. 13.

Taraborrelli L., Şenbabaoğlu Y., Wang L., Lim J., Blake K., Kljavin N., Gierke S., Scherl A., Ziai J., McNamara E., Owyong M., Rao S., Calviello A.K., Oreper D., Jhunjhunwala S., Argiles G., Bendell J., Kim T.W., Ciardiello F., Wongchenko M.J., de Sauvage F.J., de Sousa e Melo F., Yan Y., West N.R, & Murthy A. (2023). Tumor-intrinsic expression of the autophagy gene Atg16l1 suppresses anti-tumor immunity in colorectal cancer. Nature Communications, 14, 5945.

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Multiparametric Flow Cytometry Analysis

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Single-cell suspensions from spleens, lymph nodes, and thymi were incubated with anti-CD16/32 at 5 µg/ml for 20 min on ice to block Fc receptors. Cells were stained with the following antibodies: CD4 PerCP-Cy55, TCRβ PerCp-Cy55, and CD62L APC (TONBO); CD44 FITC, PD-1 PerCP-Cy55, CD69 Cy7PE, CD25 PerCP-Cy55 (Biolegend); CD4 BUV395, and CD8 BUV737 (BD); Vα2 PE (phycoerythrin), Vα2 Pacific Blue, Vα2 FITC, FR4 Cy7PE, and CD73 BV605 (eBioscience); and CD25 BV605 (Life Technology). Dead cells were excluded using the live/dead fixable Near-IR death cell stain kit (Invitrogen). Intracellular Foxp3-FITC staining was done according to the manufacturer’s instructions (Life Technology). For detection of negatively selected thymocytes, caspase 3 PE (BD) was used as previously described (Breed et al., 2019 (link)). For intracellular flow cytometry, antibodies against phosphorylated ERK^T202/Y204 (mAb 197G2), AKT^S473 (Cell Signaling) were used as previously described (Hsu et al., 2009 (link)). Antibody against Alexa 647–anti-rabbit IgG was used as a secondary antibody to detect phosphorylation of ERK and AKT.

Nguyen T.T., Wang Z.E., Shen L., Schroeder A., Eckalbar W, & Weiss A. (2021). Cbl-b deficiency prevents functional but not phenotypic T cell anergy. The Journal of Experimental Medicine, 218(7), e20202477.

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Multicolor Flow Cytometry Staining

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Splenocytes were stained according to the manufacturer’s guidelines with CD4-BUV395, CD8-BV786, CD44-APC-Cy7, KLRG-1-V450, IL-2-PE (BD Pharmingen), LIVE/DEAD Fixable Aqua Dead Cell Stain (ThermoFisher Scientific), and CD-127-PE-Cy7, CD3-BV711, Thy1.1-A700, IFN-γ-PE/Dazzle594 (BioLegend). Flow cytometry was performed using a BD LSR Fortessa Cell Analyzer (BD Biosciences, San Jose, CA). Data collected were analyzed using FlowJo Software (Tree Star, San Carlos, CA) and analyzed with Prism 6 software (GraphPad Software Inc).

Bozeman A.M., Laurie S.J., Haridas D., Wagener M.E, & Ford M.L. (2018). Transplantation Preferentially Induces a KLRG-1lo CD127hi Differentiation Program in Antigen-Specific CD8+ T Cells. Transplant immunology, 50, 34-42.

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Comprehensive Immune Cell Profiling

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The following antibodies were used for staining: CD4 BUV395, CD8α BUV737 (BD); CD5 PerCp-Cy5.5, CD25 PE or APC, CD44 FITC or Pacific Blue, CD45.2 PE, CD62L APC, CD69 APC, TCRβ Pacific Blue, Vα2 PE or Pacific Blue, CXCR5-biotin, PD-1 FITC, SLAM PE-Cy7, CD95 PE-Cy7, GL-7 FITC (Biolegend); B220 efluore780, CD8α efluore780, ZAP70 Alexa488, Strepavidin-A647 (Life Technology); GP66 tetramer PE or APC (NIH Tetramer Core Facility). The following antibodies were used for immunoblot analysis: ZAP70 (clone 1E7.2) (36 (link)); ZAP70-pTyr³¹⁹ (clone 65E4), LAT-pTyr¹⁹¹, Erk1/2, Erk1/2-pThr²⁰²/pTyr²⁰⁴, and PLCγ-pTyr¹⁸³, all from Cell Signaling Technology; anti-phosphotyrosine (4G10), PLCγ (Sigma-Aldrich), LAT (clone FL-233, Santa Cruz Biotechnology), FLAG (clone M2, Sigma-Aldrich), and GAPDH (clone 6C5, Santa Cruz Biotechnology).

Shen L., Matloubian M., Kadlecek T.A, & Weiss A. (2021). A disease-associated mutation that weakens ZAP70 autoinhibition enhances responses to weak and self ligands. Science signaling, 14(668), eabc4479.

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Multiparametric Flow Cytometry Analysis

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PBMCs (1 × 10⁶) were Fc blocked (130-059-901; Miltenyi Biotec), stained, and acquired on a BD LSR II or a BD LSRFortessa (5 (link)). For intracellular cytokine staining, 1 × 10⁶ PBMCs were cultured in the presence of the appropriate antiegn (ColI or ColV) in DMEM containing 5% FBS at 37°C. After overnight incubation, the cells were stimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 5 h, with the addition of brefeldin A (420601; BioLegend) for the last 3 h. The following Abs were used: CD3-FITC or CD3–Alexa Fluor 700 (317306/100216; BioLegend or 557943; BD Biosciences), CD11b-BV421 (562632; BD Biosciences), CD56-allophycocyanin (555518; BD Biosciences), CD14–Alexa Fluor 700 (325614; BioLegend), CD16-allophycocyanin-H7 (560195; BD Biosciences), LAIR1-PE (550811; BD Biosciences), HLA-DR–PE-Cy7 (25-9956-41; eBioscience), CD123-BV421 (306017; BioLegend), lineage-BV510 (348807; BioLegend), CD11c-allophycocyanin (559877; BD Biosciences), PE-IgG₁ κ isotype control (55749; BD Biosciences), CD196/CCR6-FITC (11-1969-42; eBioscience), CD194/CCR4-PECy7 (561034; BD Biosciences), CD4-BUV395 (563552/563790; BD Biosciences), CD183/CXCR3-allophycocyanin (550967; BD Biosciences), IFN-γ-BV421 (563376; BD Biosciences), IL-17-PE (559502; BD Biosciences), γδ TCR-FITC (118105; BioLegend). Data were analyzed using FlowJo (Treestar).

Agashe V.V., Jankowska-Gan E., Keller M., Sullivan J.A., Haynes L.D., Kernien J.F., Torrealba J.R., Roenneburg D., Dart M., Colonna M., Wilkes D.S, & Burlingham W.J. (2018). Leukocyte-Associated Ig-like Receptor 1 Inhibits Th1 Responses but Is Required for Natural and Induced Monocyte-Dependent Th17 Responses. Journal of immunology (Baltimore, Md. : 1950), 201(2), 772-781.

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Comprehensive Immune Profiling of PBMCs

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2 × 10⁶ PBMCs were stained with fixable blue dead cell stain (ThermoFisher) or Zombie NIR™ Fixable Viability Kit (Biolegend) in PBS, followed by washes and surface marker staining in Brilliant™ Stain Buffer (BD) with antibodies (Biolegend unless stated): CD3-BV786 (317330), CD4-BUV395 (BD) (563550), CD8-BV421 (301036), CD27-AF700 (356416), CD45RA-PE-Cy7 (304126), CD25-PEdazzle594 (356126), CD127-BV711 (351328), iTCR-PE (342904), CD19-AF488 (302219), IgD-BV510 (348220), HLA-DR-BV786 (307642), CD38-BV421 (356618), CD14-BV711 (301838), CD16-PEdazzle594 (302054), CD303-PerCP-Cy5.5 (354210) followed by subsequent washes and fixation in 2% PFA. Stains and washes were carried out in Biolegend cell staining buffer. Data was acquired using a BD LSRFORTESSA X-20 flow cytometer (1-2 × 10⁶ cells per sample) and analysis performed using FlowJo Single Cell Analysis Software (TreeStar). Cytometer Setup and Tracking (CS&T) (BD) beads were used to monitor cytometer performance. Application settings were applied prior to compensation to ensure that all immunophenotyping data was comparable over time. Gating strategies are shown in Supplementary Figure 1.

Robinson G.A., Waddington K.E., Coelewij L., Peng J., Naja M., Wincup C., Radziszewska A., Peckham H., Isenberg D.A., Ioannou Y., Ciurtin C., Pineda-Torra I, & Jury E.C. (2021). Increased apolipoprotein-B:A1 ratio predicts cardiometabolic risk in patients with juvenile onset SLE. EBioMedicine, 65, 103243.

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Isolation and Flow Cytometric Analysis of Murine Brain Immune Cells

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Mice were euthanized with CO₂ and perfused through the left ventricle with 20mL of PBS. Whole brain was removed and placed in 2.5 mL of digestion buffer (PBS, 5% FCS, 1mM HEPES) before finely chopping. 400U of Collagenase D (Roche) was added to the mixture, then incubated at 37°C for 30 min before adding 50 μL of 0.5M EDTA followed by a 5 min incubation. Digested tissue was mashed through a 40μm cell strainer, pelleted at 700g in a swinging bucket centrifuge, then resuspended in 10mL of 38% isotonic Percoll, and centrifuged at 2000 RPM for 30 min with no brake. The myelin debris layer was removed by aspiration and the pellet washed with PBS. Cells were then blocked with 1:100 FcX (BioLegend 156604) before a 15 min incubation with the following antibodies at a 1:200 dilution in PBS: CD45.2-FITC (eBioscience 104 cat #11-0454-82), CD4-BUV395 (BD GK1.5 cat #563790), CD11b-BV421 (BioLegend M1/70 cat #101235, CX3CR1-APC (BioLegend SA011F11 cat #149008). Cells were washed in PBS, then fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) before staining with PU.1-PE (BioLegend 7C2C34 cat #681307) or rat IgG2a-PE isotype (BioLegend RTK2758, cat #400507) for 30 minutes at room temperature. Cells were washed once, resuspended, run on a LSRFortessa cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Chung H., Parkhurst C.N., Magee E.M., Phillips D., Habibi E., Chen F., Yeung B., Waldman J., Artis D, & Regev A. (2021). Joint single-cell measurements of nuclear proteins and RNA in vivo. Nature methods, 18(10), 1204-1212.

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