Cd3 bv510 okt3

Manufactured by BioLegend

CD3-BV510 (OKT3) is a fluorescently labeled monoclonal antibody that targets the CD3 antigen on human T cells. It is a core component for the identification and analysis of T cell populations using flow cytometry.

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Lab products found in correlation

Annexin 5 percp cy5, by BioLegend (1 mentions) Anti cd19 apc cy7 sj25c1, by BD (1 mentions) Cd4 bv510, by BioLegend (1 mentions) Af933 sp, by R&D Systems (1 mentions) Live dead aqua, by Thermo Fisher Scientific (1 mentions) Igd fitc ia6 2, by BD (1 mentions) Aqua dead cell stain, by Thermo Fisher Scientific (1 mentions) Igm percp cy5.5 g20 127, by BD (1 mentions) Cd27 bv605, by BioLegend (1 mentions) Cd14 bv510 m5e2, by BioLegend (1 mentions) Facsaria 2, by BD (1 mentions)

2 protocols using cd3 bv510 okt3

SARS-CoV-2 Spike RBD Protein Characterization

Cited 1 time

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Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation and cryopreserved. SARS-CoV-2 recombinant protein was generated, briefly SARS-CoV-2 Spike RBD domain consisted of residues Thr-333 to Thr-531 (uniprot P0DT2). The RBD sequence was cloned in frame with a his8 and avi-tag sequence, respectively (RBDhisavi). RBDhisavi was expressed in insect cells and purified by nickel affinity chromatography. Purified RBDhisavi was biotinylated in vitro using BIRA (https://www.avidity.com/) for subsequent formation of RBDhisavi-tetramers for B cell staining experiments. The same RBD sequence was cloned in frame with a murine IgG1 FC domain (RBD-FC). RBD-FC was expressed in insect cells and purified by protein A affinity chromatography. Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described,⁴⁴ (link) using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Piepenbrink M.S., Park J.G., Oladunni F.S., Deshpande A., Basu M., Sarkar S., Loos A., Woo J., Lovalenti P., Sloan D., Ye C., Chiem K., Bates C.W., Burch R.E., Erdmann N.B., Goepfert P.A., Truong V.L., Walter M.R., Martinez-Sobrido L, & Kobie J.J. (2021). Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters. Cell Reports Medicine, 2(3), 100218.

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Identifying H2-Specific Memory B Cells

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Cryopreserved PBMCs from blood collected 1 and 2 weeks after the H2-F boost were stained with anti-human monoclonal antibodies CD3 BV510 (OKT3, 1:400 dilution, BioLegend, RRID:AB_2561376), CD56 BV510 (HCD56, 1:200 dilution, BioLegend, RRID:AB_2561385), CD14 BV510 (M5E2, 1:200 dilution, BioLegend, RRID:AB_2561379), CD27 BV605 (O323, 1:50, BioLegend, RRID:AB_11204431), CD20 APC-Cy7 (2H7, 1:400 dilution, BioLegend, RRID:AB_314261), IgG BV421 (G18-145, 1:50 dilution, BD Biosciences, RRID:AB_2737665), IgM PercpCy55 (G20-127, 1:40 dilution, BD Biosciences, RRID:AB_10611998), CD19 ECD (H3-119, 1:50 dilution, BD Biosciences, RRID:AB_130854), CD21 PeCy5 (B-ly4, 1:100 dilution, BD Biosciences, RRID:AB_394028) and CD38 (HIT2, 1:400 dilution, BD Biosciences, RRID:AB_1727472). H2 A/Singapore/2/1957 ectodomain and stabilized stem HA probes were expressed, biotinylated and labeled with fluorochromes as described previously (Whittle et al., 2014). Aqua dead cell stain was added for live/dead discrimination (ThermoFisher Scientific). Stained samples were run on a FACS Aria II (BD Biosciences) and data analyzed using FlowJo (TreeStar). CD3- CD14- CD56- CD19+ CD20- CD21- CD27^hi CD38^hi plasmablasts or CD3- CD14- CD56- CD19+ CD20+ IgG+ IgM- Memory B cells were gated, and H2 HA-binding B cells were single-cell sorted into 96-well plates. H2 HA head-specific B cells were identified by indexing.

Yang Y.R., Han J., Perrett H.R., Richey S.T., Jackson A.M., Rodriguez A.J., Gillespie R.A., O’Connell S., Raab J.E., Cominsky L.Y., Chopde A., Kanekiyo M., Houser K.V., Chen G.L., McDermott A.B., Andrews S.F, & Ward A.B. (2023). Immune memory shapes human polyclonal antibody responses to H2N2 vaccination. bioRxiv.

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