Anti cd3 bv650

Manufactured by BD

Sourced in United States

Anti-CD3-BV650 is a fluorochrome-conjugated antibody that binds to the CD3 cell surface antigen. It is commonly used in flow cytometry applications for the identification and analysis of T cells.

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Anti-CD3 (541 citations) CD3-BV650 (11 citations)

8 protocols using anti cd3 bv650

Multiparametric Flow Cytometric Immune Cell Profiling

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The immune cell phenotyping and analysis of functional markers were performed by multiparametric flow cytometry analysis. Briefly, cells isolated from the spleen following RBC lysis were incubated at 37°C, 5% CO₂ for 3–4 h with brefeldin A (GolgiPlug). Cells were first blocked using mouse Fc-block (anti-CD16/32) followed by staining for surface markers. Next, cells were washed, fixed, and permeabilized using the Fix-Perm reagent kit (BD Biosciences, Franklin Lakes, NJ, USA) followed by staining for intracellular markers. The following fluorochrome-conjugated anti-mouse antibodies were used: anti-CD11c APC, anti-CD19 BV605, anti-CD44 FITC, anti-CD62L APC-Cy7, anti-granzyme B BV421, anti-IFN-γ BV711, anti-IL4 PE, anti-B220 BUV737, anti-CD3 BV650, anti-CD4 BV786, anti-CD8 BUV396, anti-Foxp3 PE-CF594, and anti-GL7 PerCp-Cy5.5 (BD Biosciences). FACS data acquisition was done on a five-laser Fortessa X-20 flow cytometer (BD Biosciences) and analyzed using FlowJo version 10 (FlowJo LLC, Ashland, OR). Forward and side scatter parameters were used to set singlets and leukocyte gates. Fixable viability stain BV510 included in the surface antibody cocktail was used to gate out dead cells and analyze only viable cells. Overall gating strategy is shown (Supplemental Figure S1).

Wei J., Hegde V.L., Yanamandra A.V., O’Hara M.P., Keegan B., Jones K.M., Strych U., Bottazzi M.E., Zhan B., Sastry K.J, & Hotez P.J. (2022). Mucosal Vaccination With Recombinant Tm-WAP49 Protein Induces Protective Humoral and Cellular Immunity Against Experimental Trichuriasis in AKR Mice. Frontiers in Immunology, 13, 800295.

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Phenotypic Profiling of Immune Cells

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Cryopreserved PBMCs were first stained with fixable viability stain 510 (BD Biosciences, San Diego, CA, USA) for 15 min to exclude dead cells, then washed, and surface stained with anti-CD3-BV650 (BD Biosciences), anti-CD4-AF700 (Biolegend, San Diego, CA, USA), anti-CD8-Percp-Cy5.5 (BD Biosciences), anti-CD38-BV786 (BD Biosciences), anti-HLA-DR-Pacific blue (Biolegend), anti-CD160-AF488 (eBioscience San Diego, CA, USA), anti-PD-1-BV605 (Biolegend), anti-TIGIT-APC (eBioscience), and anti-TIM-3-PE (R&D Systems, Inc., USA) at room temperature for 20 min. Cells were permeabilized and fixed with intracellular staining reagents according to the manufacturer’s instructions (eBioscience) before intracellular staining with anti-ki67-PE-Cy7 (Biolegend). Samples were then washed before acquisition and analysis on a BD LSRFortessa flow cytometer instrument with Diva software (BD Biosciences). Relative isotype controls or fluorescence minus one samples were prepared to facilitate gating. Data were analyzed using Flowjo Software version 10 (Tree Star Inc., Ashland, OR, USA).

Xia H., Jiang W., Zhang X., Qin L., Su B., Li Z., Sun J., Zhang Y., Zhang T., Lu X, & Wu H. (2018). Elevated Level of CD4+ T Cell Immune Activation in Acutely HIV-1-Infected Stage Associates With Increased IL-2 Production and Cycling Expression, and Subsequent CD4+ T Cell Preservation. Frontiers in Immunology, 9, 616.

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Quantifying Tumor-Infiltrating Lymphocytes and Immunomodulatory Ligands

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To determine the proportion of tumor infiltrated lymphocytes in tumors after various treatments, tumor tissues harvested from the sacrificed mice were spliced into small pieces and incubated in RPMI-1640 medium comprising 1 mg/ml collagenase IV and 0.2 mg/ml DNase I at 37°C for 45 min. The solution was then filtered through a 70 μm filter to acquire single cell suspension. After the viability staining and FcγR blocking, the cell suspension was stained with Fixable Viability Stain 510 (BD Biosciences), anti-CD45-PE-Cy7 (BD Biosciences), anti-CD3-BV650 (BD Biosciences), anti-CD8-FITC (BD Biosciences), and anti-IFN-γ-APC (BD Biosciences) according to manufacturer’s protocol. Flow cytometry was performed on BD LSR Fortessa (BD Biosciences) and analyzed using FlowJo (Tree Star) software.
To determine the expression level of different immunomodulatory ligands on BMFs and tumor cells, EGFP⁺BMFs and tumor cells were harvested and incubated with Fixable Viability Stain 780 (BD Biosciences), anti-PD-L1-APC (BD Biosciences), anti-CD70-PE(BD Biosciences), anti-CTLA-4-PE(BD Biosciences), anti-CD80-PE(BD Biosciences), anti-CD86-APC(BD Biosciences), anti-MHC-I-PE-Cy7 (eBioscience)and MHC-II-PE (BioLegend) antibodies according to manufacturer’s protocol. Flow cytometry was performed on CytoFLEX device (Beckman Coulter) and analyzed using FlowJo (Tree Star) software.

Huang T., Li F., Cheng X., Wang J., Zhang W., Zhang B., Tang Y., Li Q., Zhou C, & Tu S. (2021). Wnt Inhibition Sensitizes PD-L1 Blockade Therapy by Overcoming Bone Marrow-Derived Myofibroblasts-Mediated Immune Resistance in Tumors. Frontiers in Immunology, 12, 619209.

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Phosphorylated STAT Protein and CD Marker Antibodies

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Antibodies specific to phosphorylated signal transducer and activator of transcription proteins (pSTATs) and cluster of differentiation (CD) molecules were supplied by BD Biosciences (San Jose, CA, USA): anti‐pSTAT1‐AF647 (Catalog No. 612597); anti‐pSTAT1‐AF488 (Catalog No. 612596); anti‐pSTAT3‐AF647 (Catalog No. 557815); anti‐pSTAT4‐AF647 (Catalog No. 558137); anti‐pSTAT5‐AF488 (Catalog No. 612598); anti‐pSTAT5‐AF647 (Catalog No. 612599); anti‐CD3‐BV421 (Catalog No. 562426); anti‐CD3‐BV650 (Catalog No. 563852); anti‐CD14‐Pacific Blue (Catalog. No. 558121); anti‐CD14‐AF488 (Catalog No. 557700); anti‐CD19‐BV421 (Catalog No. 562440).

Dowty M.E., Lin T.H., Jesson M.I., Hegen M., Martin D.A., Katkade V., Menon S, & Telliez J. (2019). Janus kinase inhibitors for the treatment of rheumatoid arthritis demonstrate similar profiles of in vitro cytokine receptor inhibition. Pharmacology Research & Perspectives, 7(6), e00537.

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In-Vitro Tetramer Staining Protocol

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Tetramer staining was performed on total cells after in-vitro vaccination experiments by combining 1mL of each tetramer specificity, and two different streptavidin-labelled tetramers per specificity. The staining was performed during 20 min at RT in a final volume of 100 mL of PBS 1% BSA per 1M cells. Then, 100 mL of surface antibody mix containing anti-CD3 BV650 (BD Biosciences) and anti-CD8 PECy7 (BD Biosciences) at 1/200 final dilution was added and incubated for further 20 min at 4 C. Finally, cells were washed twice with PBS-1% BSA and analyzed by flow cytometry. Live/Dead Aqua-405nm (ThermoFisher) was used to exclude dead cells. Data was collected using a ZE5 Cell Analyzer (Bio-Rad) and analyzed using FlowJo v10.3.
Tetramer analysis was done on live, single cells, CD3+CD8+ cells following the strategy described by Andersen et al. (Andersen et al., 2012) (link). Expansions were considered positive when positive for both streptavidin-labelled tetramers. Expanded populations for each peptide are represented either as frequencies of total CD8+ cells in each replicate or as total tetramer frequencies among total CD8+ T cells evaluated in all replicated for one donor.

Bonté P.E., Arribas Y.A., Merlotti A., Carrascal M., Zhang J.V., Zueva E., Binder Z.A., Alanio C., Goudot C, & Amigorena S. (2022). Single-cell RNA-seq-based proteogenomics identifies glioblastoma-specific transposable elements encoding HLA-I-presented peptides. Cell reports, 39(10).

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Phenotypic Analysis of Activated T Cells

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Cells (10 7 /ml) were plated and incubated at 37°C in an atmosphere of 5% CO 2 for 5 h in RPMI-1640 (Sigma-Aldrich, Steinheim, Germany) with penicillin-streptomycin (Sigma-Aldrich), L-glutamine (Sigma-Aldrich), and foetal bovine serum (Sigma-Aldrich) in the presence of 50 ng/ml phorbol 12-myrisate 13-acetate (Sigma-Aldrich), 250 ng/ml ionomycin (Sigma-Aldrich), and 2 μg/ml monensin (Sigma-Aldrich). Thereafter, the cells were immediately stained with the following antibodies from BD Biosciences: anti-CD3-BV650 (563852), anti-CD4-AF700 (557922), and anti-CD8-APC-H7 (560179). Next, they were fixed and permeabilized according to the manufacturer's protocol (Permeabilization Buffer 00-8333-56, Fixation/Permeabilization Diluent 00-5223-56, and Fixation/Permeabilization Concentrate 00-5123-43; eBioscience, San Diego, CA, USA). Finally, the cells were stained with the following antibodies: anti-IL5-PE (554395; BD Biosciences), anti-IL10-BV421 (564053; BD Biosciences), anti-IL17-BV510 (563295; BD Biosciences), anti-IL22-PerCP eFluor (710 46-7229-42; eBioscience), and anti-interferon (IFN)-γ-BV488 (557718; BD Biosciences). Brilliant Stain Buffer was added to the antibody cocktail.

Simonsen S., Bonefeld C.M., Thyssen J.P., Geisler C, & Skov L. (2019). Increase in Vitamin D but not Regulatory T Cells following Ultraviolet B Phototherapy of Patients with Atopic Dermatitis. Acta dermato-venereologica, 99(2).

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Flow Cytometry Analysis of PBMCs

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All FACS measurements were conducted using a FACS Celesta (BD Biosciences, Franklin Lakes, NJ, USA). PBMC were thawed rapidly at 37°C. After washing with cold PBS, cells were counted using a Neubauer counting chamber and diluted to desired concentrations. For FACS analysis, PBMC were blocked with human serum for 30 min at 4°C and incubated with a pre-titrated antibody cocktail including RTX-AF647 as well as AF750 (Thermo Fisher Scientific), for live-dead staining. For FACS analysis, in each sample at least 200,000 cells were regularly acquired. If not otherwise indicated, all antibodies were obtained from BioLegend (San Diego, CA, USA). The following antibodies and fluorophores were used: V450 anti-CD27 (clone: M-T271), V500 anti-IgD (clone: IA6-2), BV650 anti-CD3 (clone: OKT3), BV785 anti-CD45 (clone: HI30), FITC anti-CD38 (clone: HIT2; BD BioSciences), PE anti-IgM (clone: MHM-88), PerCP anti-CD4 (clone: L200), PE-Cy7 anti-CD19 (clone: HIB19), AF700 anti-CD8a (clone: HIT8a). PBMC were washed twice and measured using a FACS Celesta (BD BioSciences). Graphical analysis was performed using FlowJo version 10.6.1 (FlowJo, Ashland, OR, USA). Of note, in contour plots not all cells are depicted as single dots. For gating strategy see supplemental information (Figure S1).

Webendörfer M., Reinhard L., Stahl R.A., Wiech T., Mittrücker H.W., Harendza S, & Hoxha E. (2021). Rituximab Induces Complete Remission of Proteinuria in a Patient With Minimal Change Disease and No Detectable B Cells. Frontiers in Immunology, 11, 586012.

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T Cell Activation and Proliferation Assay

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Tissue cell suspensions were stained with 0.5 μg ml⁻¹ CFSE before activation with 10 μl αCD3/CD28 (StemCell Technologies). The cells were incubated at 37 °C for 72 h. After, the cells were stained with Live/Dead Fixable Aqua Cell Stain Kit (Thermo Fisher) for 20 min at 4 °C before being stained with BV650 anti-CD3 (BD Biosciences), BUV805 anti-CD8 (BD Biosciences), BUV395 anti-CD103 (BD Biosciences), BV421 anti-CD61 (BD Biosciences), PerCP/Cy5.5 anti-CD45RO (BioLegend), PE anti-CD49a (BD Biosciences and BioLegend) and PE/Cy7 anti-CD69 (BioLegend; details mentioned above). Following antibody staining, cells were fixed with 1× CellFix (BD Biosciences) and acquired on a BD LSR Symphony (BD Biosciences) using BD FACSDiva v9.0 software and analyzed on FlowJo v.10.5.3 (TreeStar). Cells were considered proliferative based on the decrease in CFSE fluorescence, within the 1st downward peaks of CFSE onwards.

Hamid M.H., Cespedes P.F., Jin C., Chen J.L., Gileadi U., Antoun E., Liang Z., Gao F., Teague R., Manoharan N., Maldonado-Perez D., Khalid-Alham N., Cerundolo L., Ciaoca R., Hester S.S., Pinto-Fernández A., Draganov S.D., Vendrell I., Liu G., Yao X., Kvalvaag A., Dominey-Foy D.C., Nanayakkara C., Kanellakis N., Chen Y.L., Waugh C., Clark S.A., Clark K., Sopp P., Rahman N.M., Verrill C., Kessler B.M., Ogg G., Fernandes R.A., Fisher R., Peng Y., Dustin M.L, & Dong T. (2024). Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity. Nature Immunology, 25(5), 834-846.

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