Cd8 bv510

Manufactured by BioLegend

Sourced in United States

CD8-BV510 is a fluorescent-conjugated antibody used to identify and quantify CD8-positive cells in flow cytometry applications. It targets the CD8 surface marker, which is expressed on cytotoxic T cells and a subset of natural killer cells.

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Close spelling variants of this product

Anti-CD8-BV510 (10 citations) CD8 BV510 (RPA-T8; (5 citations) CD8-BV510 (clone SK1, (4 citations) CD8 BV510 (RPA-8, (3 citations) Anti-human CD8 BV510 (2 citations) BV510-anti-CD8 (53-6.7; (2 citations) Anti-mouse CD8 BV510 (2 citations) BV510-antimouse CD8 (2 citations)

32 protocols using cd8 bv510

NK cell CD107A and T cell Cytokine Assay

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For detection of CD107A expression on NK cells, we resuscitated PBMCs frozen from patients and healthy controls and cultured them in a 37 ℃, 5% CO₂ incubator for 2 h. Then, the stimulation group was co-cultured with K562 cells for 4 h. The cells were taken out for staining with CD3-Percp (300,326 Biolegend), CD56-PE (318,306 Biolegend), and CD107a-APC (328,620 Biolegend) at room temperature for 30 min. Then, the cell was analyzed by flow cytometer. For detection of IL-17A/IFN production, the frozen-stored PBMC of patients and healthy controls were resuscitated and cultured in a 37 ℃, 5% CO₂ incubator for 4 h. Then, the stimulation group was activated with 1 μg/ml PMA (P8139 Sigma Aldrich), ionomycin (Inhion Invivogen) and Golgi stop (554,724 BD) for 4 h. The cells were taken out and incubated with CD3-Percp-cy5.5 (317,336 Biolegend) and CD8-BV510 (344,732 Biolegend), 4 ℃ for 30 min. After taking out the cells, they were fixed and punched (BD), and then stained with CD17A-PE (512,306 Biolegend) and IFN-γ-APC (502,512 Biolegend) for 1 h, then analyzed by flow cytometer.

Li W., Sun Y., Yu L., Chen R., Gan R., Qiu L., Sun G., Chen J., Zhou L., Ding Y., Du H., Shu Z., Zhang Z., Tang X., Chen Y., Zhao X., Zhao Q, & An Y. (2023). Multiple Immune Defects in Two Patients with Novel DOCK2 Mutations Result in Recurrent Multiple Infection Including Live Attenuated Virus Vaccine. Journal of Clinical Immunology.

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Intracellular Cytokine Staining of Th17 and Th1 Cells

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PBMCs were isolated by density gradient centrifugation using Histopaque (Sigma, Dorset, UK), frozen and stored in liquid nitrogen before staining. Intracellular cytokine staining of Th17 and Th1-cells was carried out using BD Cytofix/Cytoperm kit (BD Bioscience, Oxford, UK). Cells were stimulated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma, Dorset, UK) and 1 μg/mL ionomycin (Sigma, Dorset, UK) for 4 h in the presence of Golgi STOP and Golgi plug. After surface staining using CD3-BV605, CD4-APC and CD8-BV510 antibodies (Biolegend, London, UK), cells were fixed and permeabilised, then stained with IL-17A-FITC (eBiosciences, Ireland, UK) and interferon (IFN)-γ-AF700 (Biolegend). Dead cells were excluded using Fixable Viability Dye eFluor 780 (eBiosciences). Representative FACS plots of the gating strategy and intracellular staining are shown in online supplementary figure S1.

Roberts A.R., Vecellio M., Chen L., Ridley A., Cortes A., Knight J.C., Bowness P., Cohen C.J, & Wordsworth B.P. (2016). An ankylosing spondylitis-associated genetic variant in the IL23R-IL12RB2 intergenic region modulates enhancer activity and is associated with increased Th1-cell differentiation. Annals of the Rheumatic Diseases, 75(12), 2150-2156.

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Multicolor Flow Cytometry for T and B Cell Subsets

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PBMC were isolated from peripheral blood by Ficoll gradient and frozen for batched analysis. We designed six multicolor flow cytometry panels to quantify 60 T cell subsets along with two B cell subsets, and calculated the CD4⁺/CD8⁺ T cell ratio (Supplementary Table 1). The following fluorochrome-conjugated anti-human antibodies were used from BD Biosciences (San Jose, CA): CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CCR4-PE, CD27-PE, CD28-BV421, CD138-BV421, CCR6-BV421, CXCR3-PE, CCR7-A700, IL-17-BV786, IFN-γ-PE-Cy7, iso IgG1k-FITC, iso IgG1k-PE-Cy7, iso IgG2bk-APC, iso IgG1k-APC-Cy7, and iso IgG1k-BV510; from Biolegend (San Diego, CA): CD127-FITC, CD27-APC, CD57-PerCp-Cy5.5, CD19-BV510, PD-1-APC-Cy7, CXCR5-FITC, and TNFα-FITC; from eBioscience (San Diego, CA): CD4-PE-Cy7, and IL-2-PE; from Miltenyi Biotec (San Diego, CA): CD25-APC and KLRG1-PE; from Beckman Coulter (Brea, CA): CD38-PE-Cy7.

Hartzell S., Bin S., Cantarelli C., Haverly M., Manrique J., Angeletti A., Manna G.L., Murphy B., Zhang W., Levitsky J., Gallon L., Yu S.M, & Cravedi P. (2020). Kidney Failure Associates With T Cell Exhaustion and Imbalanced Follicular Helper T Cells. Frontiers in Immunology, 11, 583702.

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Multiparametric Flow Cytometry of Tumor Cells

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Single-cell suspensions of tumors were prepared, and flow cytometry was carried out as mentioned earlier [45 (link), 46 (link)]. Cells were stained with the following antibodies obtained from BioLegend: CD16/32 (clone 93, catalog No. 103132), Zombie UV™ Fixable Viability Kit (catalog No. 100752), CD45-PerCP/Cy5.5 (clone 30-F11, catalog No. 103132), CD45-APC/Cy7 (clone 30-F11, catalog No. 103116), CD11b-FITC (clone M1/70, catalog No. 101206), SIRPα-PE (clone BM8, catalog No. 123122), Gr-1-PerCP/Cy5.5 (clone RB6-8C5, catalog No. 108428), F4/80-AF647 (clone N418, catalog No. 117318),CD11c-PE/Cy7 (clone N418, catalog No. 117318), I-A/I-E-AF700 (clone M5/114.15.2, catalog No. 107622), CD86-BV421 (clone GL-1, catalog No. 105032), CD206-BV605 (clone C068C2, catalog No. 141721), CD8a-AF700 (clone 53-6.7, catalog No. 100730), CD8-BV510 (clone 53-6.7, catalog No. 100752), TNF-α-BV421 (clone MP6-XT22, catalog No. 506328), IFN-γ-PE (clone XMG1.2, catalog No. 505808), CD39-PE/Cy7 (clone Duha59, catalog No. 143806), Tim-3-APC/Fire™ 750 (clone RMT3-23, catalog No. 119738), PD-1-BV605 (clone 29 F.1A12, catalog No. 135220). The next antibodies were obtained through eBioscience: Mouse Regulatory T Cell Staining Kit #2 (clone FJK-16s, catalog No. 88-8118-40). FlowJo program (Tree Star Inc.) was utilized to examine the findings that were acquired through a BD LSRFortessa Flow Cytometer.

Ji K., Zhang Y., Jiang S., Sun L., Zhang B., Hu D., Wang J., Zhao L., Wang P, & Tao Z. (2023). SIRPα blockade improves the antitumor immunity of radiotherapy in colorectal cancer. Cell Death Discovery, 9, 180.

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Multiparameter Flow Cytometry of Immune Cells

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Mouse cells were stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly-conjugated monoclonal antibodies to CD45-PE-Dazzle594 (Biolegend, 104), TCRβ-APCef780 or APC (Invitrogen, H57-597), CD8-BV510 (Biolegend, 53-6.7), then fixed, permeabilized and stained for the cytokines IL-17A-PerCpCy5.5 or PE (e-Biosciences, 17B7), IFNγ-PE-Cy7 (e-Biosciences, XMG1.2) and TNFα-BV650 (Biolegend, MP6-XT22).

Oates J.R., Sawada K., Giles D.A., Alarcon P.C., Damen M.S., Szabo S., Stankiewicz T.E., Moreno-Fernandez M.E, & Divanovic S. (2023). Thermoneutral housing shapes hepatic inflammation and damage in mouse models of non-alcoholic fatty liver disease. Frontiers in Immunology, 14, 1095132.

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Multiparameter Flow Cytometry of PBMCs

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PBMCs were incubated with directly conjugated antibodies for 30 min at 4°C. The cells were then washed before flow cytometric analysis. Antibodies used were anti-human CD3-BUV737, CD4-BUV395, PD-1-BV711, CD38-FITC, GITR-BV605 (BD Biosciences, San Diego, CA, USA), CD8-BV510, CTLA-4-BV786, OX40-APC-Fire750, 4-1BB-BV421, HLA-DR-AF700 (BioLegend, San Diego, CA, USA), TIGIT- PE-Cy7, LAG-3-APC, ICOS-PE, (Ebioscience, San Diego, CA, USA), and the corresponding isotype controls. Data acquisition was performed on an LSR Fortessa flow cytometer (BD Biosciences), and data analysis was performed using FlowJo Software (Tree Star, Ashland, OR, USA).

Du J., Wei L., Li G., Hua M., Sun Y., Wang D., Han K., Yan Y., Song C., Song R., Zhang H., Han J., Liu J, & Kong Y. (2021). Persistent High Percentage of HLA-DR+CD38high CD8+ T Cells Associated With Immune Disorder and Disease Severity of COVID-19. Frontiers in Immunology, 12, 735125.

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Phenotyping PfCSP+ Memory B Cells

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Cryopreserved PBMCs from the VRC 314 trial were thawed and stained for 30 minutes at room temperature in the dark with the following panel: 1:500 Blue LIVE/DEAD (Thermo Fisher Scientific L23105), 18 μg/mL IgA-Dylight 405 (Jackson Immunoresearch 109-475-011), 1:50 CD14-BV510 (BioLegend 301842), 1:50 CD3-BV510 (BioLegend 317332), 1:100 CD8-BV510 (301048), 1:50 CD56-BV510 (BioLegend 318340), 1:50 CD27-BV605 (BioLegend 302830), 1:40 CD21-BV711 (BD Biosciences 563163), 1:50 CD19-BV750 (BioLegend 302236), 1:125 IgD-PECy7 (BD Biosciences 561314), 1:20 IgM-Brilliant Ultraviolet (BUV)395 (BD Biosciences 563903), 1:125 CD38-Alexa Fluor 700 (BD Biosciences 560676), 1:40 IgG-allophycocyanin (APC)H7 (BD Biosciences 561297), 1:25 PfCSP-Brilliant Blue (BB)660, and 1:25 PfCSP-BUV737. The PfCSP probes were made by conjugating biotinylated PfCSP to streptavidin tagged with the relevant fluorescent dye. The cells were sorted using a BD FACS Aria II and analyzed with FlowJo software (Tree Star). Cells were gated on live CD19⁺CD14^-CD3^-CD8^-CD56^-CD21⁺CD27⁺IgA⁺/IgG⁺, with CD27⁺⁺CD38⁺⁺ cells excluded.

Tan J., Cho H., Pholcharee T., Pereira L.S., Doumbo S., Doumtabe D., Flynn B.J., Schön A., Kanatani S., Aylor S.O., Oyen D., Vistein R., Wang L., Dillon M., Skinner J., Peterson M., Li S., Idris A.H., Molina-Cruz A., Zhao M., Olano L.R., Lee P.J., Roth A., Sinnis P., Barillas-Mury C., Kayentao K., Ongoiba A., Francica J.R., Traore B., Wilson I.A., Seder R.A, & Crompton P.D. (2021). Functional human IgA targets a conserved site on malaria sporozoites. Science translational medicine, 13(599), eabg2344.

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Multiparameter Flow Cytometry of PBMCs

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PBMCs and direct coupling antibody were incubated together in dark environment at 4°C for 20 minutes. Then 2 mL of 1 × PBS were added to wash lymphocytes. After rotating 1200 rpm for 5 minutes, the supernatant was discarded. Finally, 200 mL of 1 × PBS was added to make the pre-test sample. The monoclonal antibodies were anti-human CD4-APC-Fire750 (Clone SK3), CD8-BV510 (Clone SK1), HLA-DR-PE-Cy7 (Clone L243), and CD38^-APC (Clone HB-7) (BioLegend, San Diego, CA, USA). FACSCanto flow cytometry (BD Biosciences, San Diego, CA, USA) and FlowJo software (Tree Star, Ashland, OR, USA) was used to gather and analyze the data, respectively.

Lin Y., Shen G., Xie S., Bi X., Lu H., Yang L., Jiang T., Deng W., Wang S., Zhang L., Lu Y., Gao Y., Hao H., Wu S., Liu R., Chang M., Xu M., Hu L., Chen X., Huang R., Li M, & Xie Y. (2023). Dynamic changes of the proportion of HLA-DR and CD38 coexpression subsets on T lymphocytes during IFN-based chronic hepatitis B treatment. Frontiers in Immunology, 13, 1116160.

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Multi-parameter Flow Cytometry Assay

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Anti-Bcl-2 PE, CD3-FITC, CD8-APC, CD127-BV510, IFNγ-APC, RORγt-BV421, TNF-PE were from BD Biosciences. Anti-CD4-PerCP, CD8-BV510, CD127-PECy7, CD161-BV421, -FITC, and -PE, IFNγ-APC, Ki-67-AF488 and -BV510, TNF-BV421, and Vα7.2 PECy7 were from Biolegend. Anti-PLZF APC was from R&D Systems. CellTrace Violet (CTV) cell proliferation kit, Live/Dead Aqua and Near Infrared fixable cell stain were from Life Technologies. MR1-5-OP-RU tetramer -APC, -BV421, and -PE were from NIH Tetramer Core Facility, Emory University. Staining with the MR1 5-OP-RU tetramers was performed for 40 min at room temperature (RT) (6 (link)) before proceeding to the surface and intracellular staining with other mAbs. Cell surface and intracellular staining for TFs, cytokines, and cytotoxic molecules were performed as previously described (10 (link)). Samples were acquired on an FACS Canto II (BD Biosciences) equipped with 405, 488, and 633 nm lasers or CytoFLEX (Beckman Coulter) equipped with 405, 488, and 638 nm lasers. Data including the compensation platform were analysed using FlowJo v.10 (BD Biosciences).

Han F., Gulam M.Y., Zheng Y., Zulhaimi N.S., Sia W.R., He D., Ho A., Hadadi L., Liu Z., Qin P., Lobie P.E., Kamarulzaman A., Wang L.F., Sandberg J.K., Lewin S.R., Rajasuriar R, & Leeansyah E. (2022). IL7RA single nucleotide polymorphisms are associated with the size and function of the MAIT cell population in treated HIV-1 infection. Frontiers in Immunology, 13, 985385.

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Comprehensive T Cell Activation Profiling

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Peptide stimulated cells were washed and extracellularly stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Afterwards, samples were fixed and permeabilized for 30 min at 4°C using FoxP3 transcription factor staining buffer set (eBioscience). Following, intracellular staining was performed for CD3 BV650, CD4 PerCp-Cy5.5, CD8 BV510, CD137 PE, CD154 BV421, IL-2 APC, IFNγ BV605, and TNFα AF700 (Biolegend). Stained cells were then transferred into a 96-well plate and measured at a CytoflexLX (Beckman Coulter). Flow cytometry data was analyzed using FlowJo software version 10.6.2 (BD). Reactive T cells were defined as CD154+CD137+CD4+ or CD137+CD8+ T cells >0.005% within total CD4+ or CD8+ T cells and with a threshold of ≥1.2-fold signal above the background control. This threshold corresponds to the range in which 95% of all negative samples are. Unspecific activation of cells was excluded by subtracting the background signal of the DMSO stimulated negative control sample from the peptide stimulated samples. Single, double (dp), or triple (tp) cytokine producing reactive T cell subsets were analyzed using Boolean combination gates (see Supplementary Figure 1 for gating strategy).

Steiner S., Schwarz T., Corman V.M., Sotzny F., Bauer S., Drosten C., Volk H.D., Scheibenbogen C, & Hanitsch L.G. (2021). Reactive T Cells in Convalescent COVID-19 Patients With Negative SARS-CoV-2 Antibody Serology. Frontiers in Immunology, 12, 687449.

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