Cd19 bv605

Manufactured by BioLegend

Sourced in United States

The CD19-BV605 is a fluorescently-labeled antibody that binds to the human CD19 cell surface antigen. It can be used for the identification and enumeration of CD19-positive cells in flow cytometry applications.

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Lab products found in correlation

Cd11b percp cy5, by BioLegend (5 mentions) Cd3 fitc, by BioLegend (3 mentions) Cd3 apc cy7, by BioLegend (3 mentions) Lsrfortessa, by BD (3 mentions) Cd45 buv395, by BD (3 mentions) Ly6c pacific blue, by BioLegend (3 mentions) Lsrfortessa flow cytometer, by BD (2 mentions) Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (2 mentions) Cd4 fitc, by BioLegend (2 mentions) Cd3 bv421, by BioLegend (2 mentions) Dnase 1, by Roche (2 mentions) Cd3 apc, by BioLegend (2 mentions) Fortessa x20, by BD (2 mentions) Ly6g pe cy7, by BioLegend (2 mentions) Macsquant analyzer, by Miltenyi Biotec (2 mentions) Ethylene diamine tetraacetic acid (edta), by Corning (2 mentions) Cd16 fitc, by BioLegend (2 mentions) Nk1.1 pe cy7, by BioLegend (2 mentions) Ly6b fitc, by Abcam (2 mentions) Mhc class 2 a700, by BioLegend (2 mentions)

Spelling variants of this product

Anti-CD19-BV605 (6 citations) CD19-BV605 (6D5, (2 citations)

19 protocols using cd19 bv605

Multiparametric Flow Cytometry of Immune Cells

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The isolation of mononuclear cells (MNCs) was performed as described in the previous report [6 (link)]. 1 × 10⁶ MNCs were incubated with anti-CD16/CD32 antibody (BioLegend, San Diego, CA) and then stained with antibody cocktails including APC/Cy7-CD45.2 (BioLegend), BV605-CD19 (BioLegend), FITC-CD3 (BioLegend), PE/Cy7-NK1.1 (BioLegend), BUV563-CD4 (BD Biosciences, San Diego, CA), BV711-CD8a (BioLegend), BUV737-CD62L (BD Biosciences), APC-CD44 (BioLegend), PerCP/Cy5.5-GL7 (BioLegend), Alexa Fluor 488-Fas (eBioscience), and BV510-B220 (BioLegend). Flow cytometry was performed with the LSRFortessa flow cytometer (BD Immunocytometry Systems, San Jose, CA), and data were analyzed with FlowJo software (BD Immunocytometry Systems). The full gating strategy is shown in supporting information (Supplementary Figure 4).

Xu Y.F., Yao Y., Ma M., Yang S.H., Jiang P., Wang J., Tsuneyama K., Wang C., Liu X., Li L, & Lian Z.X. (2022). The Proinflammatory Cytokines IL-18, IL-21, and IFN-γ Differentially Regulate Liver Inflammation and Anti-Mitochondrial Antibody Level in a Murine Model of Primary Biliary Cholangitis. Journal of Immunology Research, 2022, 7111445.

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Multiparametric Flow Cytometry Analysis of Murine Splenocytes

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An LSR Fortessa (BD Biosciences, Franklin Lakes, NJ) was used for multiparametric flow cytometry analysis. A Live/Dead Fixable Blue Dead Cell Stain Kit (Molecular Probes; Thermo Fisher Scientific, Grand Island, NY) was used to exclude dead cells. The following murine monoclonal antibodies (mAbs) were used to stain Balb/c splenocytes to characterize immune cell subsets: FITC-CD8 (Clone 53-6.7; BD Biosciences), PE-CD49b (Clone DX5; BioLegend, San Diego, CA), PE-PD-L1 (Clone 10F.9G2; BioLegend), PerCP-Cy5.5-B7-1 (Clone 16-10A1; BioLegend), PerCP-Cy5.5-FOXP3 (Clone FJK-16s; eBioscience, San Diego, CA), PE-Cy7-CD122 (Clone Tm-b1; eBioscience), APC-NKG2D (Clone CX5; BioLegend), APC-Cy7-CD3 (Clone 17A2; BioLegend), Pacific Blue-CD44 (Clone IM7; BioLegend), AF700-CD4 (Clone GK1.5; eBioscience), BV605-CD19 (Clone 6D5; BioLegend), BV605-PD-1 (Clone 29F.1A12; BioLegend), BV510-NKp46 (Clone 29A1.4; BioLegend), Pacific Blue-CD27 (Clone LG.3A10; BioLegend), FITC-CD11b (Clone M1/70; BD Biosciences), AF700-CD11b (Clone M1/70; BioLegend), APC-Cy7-CD11c (Clone N418; BioLegend), BV510-CD3 (Clone 17A2; BioLegend), Pacific Blue-Ly6-G (Clone 1A8; BioLegend), BV605-Ly6-C (Clone HK1.4; BioLegend).

Kim P.S., Kwilas A.R., Xu W., Alter S., Jeng E.K., Wong H.C., Schlom J, & Hodge J.W. (2016). IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity against murine breast and colon carcinomas. Oncotarget, 7(13), 16130-16145.

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Immune cell profiling of quadriceps muscle

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Perfused ipsilateral quadriceps muscles were minced and digested in RPMI supplemented with 10% HI-FBS, HEPES pH 7.3, 100 U/ml penicillin and streptomycin, collagenase (2.5 mg/ml; Sigma), and DNase I (30 μg/ml; Roche) in a total of 5 ml at 37°C for 1.5 h. Digested tissue was pressed through a 70 μm cell strainer, and resuspended in RPMI supplemented with 10% HI-FBS and penicillin and streptomycin. Cells were counted using precision count beads (BioLegend). Single cell suspensions were blocked for FcγR binding (BioLegend; clone S17011E) and stained with the following antibodies: CD3 BV421 (BioLegend; clone 145-2C11), CD4 FITC (BioLegend; clone RM4-5), CD8α APC (BioLegend; clone 53–6.3), NK1.1 PE (BioLegend; clone PK136), CD45 BUV395 (BD Biosciences; clone 30-F11), CD19 BV605 (BioLegend; clone 6D5), CD11b PerCP-Cy5.5 (BioLegend; clone M1/70), Ly6C Pacific Blue (BioLegend; clone HK1.4), Ly6G phycoerythrin (PE)-Cy7 (BioLegend; clone 1A8), MHC class II Alexa Fluor 700 (BioLegend; clone M5/114.15.2), Ly6B FITC (BioLegend; clone 7/4), and F4/80 BV650 (BioLegend; clone BM8). Viability was determined by exclusion of a fixable viability dye (eBiosciences; e506). Samples were run on a BD LSRFortessa X-20 flow cytometer and analyzed using FlowJo version 10 (FlowJo, LLC).

Fox J.M., Huang L., Tahan S., Powell L.A., Crowe JE J.r., Wang D, & Diamond M.S. (2020). A cross-reactive antibody protects against Ross River virus musculoskeletal disease despite rapid neutralization escape in mice. PLoS Pathogens, 16(8), e1008743.

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Multiparametric Immunophenotyping of Hematopoietic Cells

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Anti- mouse Ter119-BV421, Sca-1-APC, CD34-FITC, CD135-PE, IL-7Ra-BV605, CD48-FITC, CD150-PE Cy7, CD45.2-Alexa Fluor 700/APC Cy7, CD11b-BV605, CD3-APC, NK1.1-APC Cy7, CD19-BV605, IgM-Alexa Fluor 647, CD43-PE Cy7, CD16/32-PerCP Cy5.5, CD16/32; anti-human CD34-APC Cy7 (clone: 581), CD38-PE Cy7 (HB-7), CD45RA-BV 605 (HI100), CD90-APC (5E10), CD49f-PerCP Cy5.5 (GoH3), CD45-PE/Dazzle 594 (HI30), and CD33-APC (P67.6) antibodies; and Legendplex™ Mouse Inflammation Panel (13-plex) and Mouse IL-6 ELISA were purchased from Biolegend (San Diego, CA). IL6 depleting AB (clone MP5–20F3) from BioXCell (West Lebanon, NH).
Anti-mouse c-Kit-PE Cy7, Mouse Hematopoietic Lineage Biotin Panel, and anti-human CD19-Alexa Fluor 700 (HIB19) from eBioscience (San Diego, CA). Anti-B220-PE CF594 from BD biosciences (San Jose, CA). EasySep Human Progenitor Cell Enrichment Kit from Stem Cell Technologies Inc. (Vancouver, BC). Anti-human EPCR-FITC (RCR-49) from Santa Cruz Biotechnology®, inc. (Dallas, TX). Anti-human CD141-PE (AD5–14H12) from Miltenyi Biotec Inc. (Auburn, CA).

Basu S., Liang H.P., Hernandez I., Zogg M., Fields B., May J., Ogoti Y., Wyseure T., Mosnier L.O., Burns R.T., Carlson K, & Weiler H. (2019). Role of Thrombomodulin expression on hematopoietic stem cells. Journal of thrombosis and haemostasis : JTH, 18(1), 123-135.

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Isolation and Staining of Myeloid Cells

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For cell depletion experiments, whole blood was harvested from mice and mixed with 5 mM EDTA (Corning). Red blood cells were lysed with ACK lysis buffer at room temperature before being washed and chilled in PBS + 5% FCS. Monocytes were stained with CD45 BUV395 (BD Biosciences clone 30-F11), CD11b PerCP-Cy5.5 (BioLegend clone M1/70), Ly6B FITC (Abcam clone 7/4), Ly6G PE-Cy7 (BioLegend clone 6D5), Ly6C Pacific Blue (BioLegend clone HK1.4) and MHC class II A700 (BioLegend clone M5/114.15.2). NK cells were stained with CD45 BUV95, CD3 APC-Cy-7 (BioLegend clone 145–2C11), CD19 BV605 (BioLegend clone 6D5), and NK1.1 Pe-Cy7 (BioLegend clone PK136). Viability was determined through exclusion of a fixable viability dye (e506;eBiosciences). Samples were fixed and processed on a BD-Fortessa X20. For FcγR expression experiments BV2, LADMAC, and Vero cells were stained with one of the following: CD64 APC (BioLegend clone X54–5/7.1), CD32b APC (Invitrogen clone AT130–2), CD16 FITC (BioLegend clone 221–3A4), CD16.2 APC (BioLegend clone 9E9). Cells were analyzed on a MACSQuant analyzer (Miltenyi). All FACS data were analyzed by FlowJo v. 10.7.

Earnest J.T., Holmes A.C., Basore K., Mack M., Fremont D.H, & Diamond M.S. (2021). The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies. Cell reports, 35(1), 108962.

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Comprehensive Tumor and Immune Cell Analysis

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SA1/N and B16-SIY tumor analyses were performed 2 days following the second dose of antibody treatment. Tumors were weighed on a microscale on plastic weigh boats and dissociated with a 0.25mg/mL Liberase TL (Sigma-Aldrich), 0.05mg/mL DNaseI (Roche) from bovine pancreas, and RPMI Medium mixture for 20 min at 37To determine ICOS receptor quantity on primary cells, tumors were processed as described above. Peripheral blood was collected by cardiac puncture with 25-gauge needles and tuberculin syringes and dispersed into EDTA coated blood collection tubes. RBCs were lysed in conical tubes with 1mL of ACK lysis buffer for 3min on ice. Blood was centrifuged at 400xg for 5mins and resuspended in 100uL FACS buffer. Tumors and peripheral blood were transferred to round bottom 96-well plates and Fc blocked as described above. Following Fc block, cells were stained with 1ug/mL DyLight-650 (Life Technologies) labelled ICOS antibody 37A10 or isotype control, in addition to the staining cocktail described above. Cells were stained in 100uL of cocktail for 30mins at 4ºC. Cells were then washed twice with FACS buffer and resuspended 100uL in Fix/Perm buffer (Foxp3/Transcription Factor Staining Kit, eBioscience) and kept at 4ºC for 15mins. Cells were washed twice in Perm buffer and resuspended in 100uL of intracellular staining cocktail containing fluorescent labelled anti-FoxP3 (FJK-16s, 1:100, eBioscience) diluted in Perm buffer. Following a 30min incubation at 4ºC, cells were washed twice in FACS buffer and resuspended in 100uL of FACS buffer supplemented with 0.1% paraformaldehyde. Receptor quantitation was performed using Quantum Simply Cellular beads (Bangs Laboratories) according to manufacturer’s protocol. To determine the number of cells per milligram of tumor, a fixed number of CountBrite beads (Life Technologies) were added to samples and analyzed by flow cytometry.
For TIL analysis of MC38 tumors, huCTLA-4 mice were randomized in groups when tumor reached an average size of 153mm³ and treated with a single dose of clinical grade ipilimumab (Yervoy®, Bristol-Meyers Squibb) or human IgG1 isotype control administered intravenously at 10 mg/kg, or a single dose of ICOS antibody or mouse IgG2a isotype control administered intraperitoneally at 0.25 mg.kg. Mice were sacrificed 72 hours later and tumors excised, dissociated, and processed for flow cytometry analysis. The following antibodies were used: CD45-BB515 (clone 30-F11, BD), CD3-BUV395 (clone 145-2C11, BD), CD4-APCeF780 (clone GK1.5, eBiosciences), CD8-PEeF610 (clone 53–6.7, eBiosciences), FoxP3-PE (clone NRRF-30, eBiosciences), CD25-PerCP/Cy5.5 (clone PC61, Biolegend), CD19-BV605 (clone 6D5, Biolegend), CD49b-APC (clone DX5, Biolegend), ICOS-BV421 (clone C398.4A, Biolegend). Viability was assessed by staining with Fixable Viability Dye eF506 (Biolegend).

Hanson A., Elpek K., Duong E., Shallberg L., Fan M., Johnson C., Wallace M., Mabry G.R., Sazinsky S., Pepper L., Shu C.J., Sathyanarayanan S., Zuerndorfer S., Simpson T., Gostissa M., Briskin M., Law D., Michaelson J, & Harvey C.J. (2020). ICOS agonism by JTX-2011 (vopratelimab) requires initial T cell priming and Fc cross-linking for optimal T cell activation and anti-tumor immunity in preclinical models. PLoS ONE, 15(9), e0239595.

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Murine Lymphoid Tissue Immunophenotyping

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Mesenteric lymph nodes and spleen samples were washed and conserved in complete RPMI (10 % FCS). Samples were first grinded on a nylon mesh (40 μm cell strainer, Greiner), and large debris was removed. Cell suspensions were filtered through a 70 μm mesh (Miltenyi Biotec) and centrifuged at 4 °C, 600 xg, for 5 min. Cell suspensions were then washed and resuspended in phosphate-buffered saline (PBS) supplemented with 2% bovine serum albumin (BSA) for counting on an automated cell before direct cell surface staining. For intracellular staining, cells were fixed and permeabilized with a commercially available fixation/permeabilization kit (eBioscience). Single-cell suspensions were stained with antibodies to the following markers: CD45-BV421, CD3-APC/Fire750, CD4-FITC, CD25-BV510, CD8a-BV711, CD335-PECy7, CD19-BV605, CD11b-PERcpCY5.5, RORγt-APC, and Foxp3-PE in presence of FCBlock CD16/32; all antibodies were obtained from Biolegend. The gating strategy is detailed in Supplementary Figure 8. Gallios cytometer (Beckman) was used for cell acquisition, and the flow cytometry data were analyzed with Kaluza software.

Arnone D., Vallier M., Hergalant S., Chabot C., Ndiaye N.C., Moulin D., Aignatoaei A.M., Alberto J.M., Louis H., Boulard O., Mayeur C., Dreumont N., Peuker K., Strigli A., Zeissig S., Hansmannel F., Chamaillard M., Kökten T, & Peyrin-Biroulet L. (2021). Long-Term Overconsumption of Fat and Sugar Causes a Partially Reversible Pre-inflammatory Bowel Disease State. Frontiers in Nutrition, 8, 758518.

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NK Cell Activation by Dendritic Cells

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NK cells were enriched from PBMCs by the depletion of unwanted cells using biotinylated antibodies and MoJo Streptavidin Nanobeads (BioLegend); more details are available in SI Appendix. The enriched NK cells were stained with CD3-BV605 (clone: UCHT-1, BioLegend), CD19-BV605 (clone: SJ25C1, BioLegend), CD20-BV605 (clone: 2H7, BioLegend), HLA-DR-BV510 (clone: L243, BioLegend), CD56-BV421 (clone: 5.1H11, BioLegend) and NKp46-BV421 (clone: 9E2, BioLegend) and sorted as CD56⁺NKp46⁺CD3⁻CD19⁻CD20⁻HLA-DR⁻ cells. Sorted XCR1⁻ and XCR1⁺ cDC1 and NK cells were cocultured in a 1:5 ratio and either stimulated with a cocktail of pIC (2.5 µg/mL), R848 (2.5 µg/mL), and CpG (2.5 µg/mL) or cultured in DC-medium alone. For some experiments, DCs and NK cells were separated in Transwell plates (96 well, 0.4 µm insert). NK cells were cultured in the well, while DCs were added to insert. In order to analyze the influence of secreted cytokines on NK cells, NK cells were cultured alone and supernatants of stimulated DCs were added corresponding to a 1:5 DC:NK cell ratio. After 18 h, cells were harvested and analyzed by flow cytometry for activation of NK cells (CD69). The supernatants were stored at −80 °C until analysis with LEGENDplex Human Interferon Panel (BioLegend) for secretion of IFNγ.

Heger L., Hatscher L., Liang C., Lehmann C.H., Amon L., Lühr J.J., Kaszubowski T., Nzirorera R., Schaft N., Dörrie J., Irrgang P., Tenbusch M., Kunz M., Socher E., Autenrieth S.E., Purbojo A., Sirbu H., Hartmann A., Alexiou C., Cesnjevar R, & Dudziak D. (2023). XCR1 expression distinguishes human conventional dendritic cell type 1 with full effector functions from their immediate precursors. Proceedings of the National Academy of Sciences of the United States of America, 120(33), e2300343120.

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Isolation and Staining of Myeloid Cells

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Earnest J.T., Holmes A.C., Basore K., Mack M., Fremont D.H, & Diamond M.S. (2021). The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies. Cell reports, 35(1), 108962.

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Comprehensive B Cell and Immune Profiling

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Flow-cytometric analysis or cell sorting of single cell suspensions generated from spleen or bone marrow was performed on an LSR-Fortessa or a FACS-Aria-III, respectively (both BD) and analyzed using FlowJo v10 software. Antibodies used (40 µl/sample): B220-FITC (biolegend RA3-6B2) 1:300, B220-APC/eF780 (ebioscience RA3-6B2) 1:200, B220-PE (BD RA3-6B2) 1:300, B220-PErCP-Cy5.5 (biolegend RA3-6B2) 1:300, CD19-BV605 (biolegend 6D5) 1:400, IgM-APC (biolegend RMM-1) 1:300, IgM-FITC (BD II/41) 1:300, IgM-eF450 (ebioscience eb121-15F9) 1:200, IgD-PerCP-Cy5.5 (biolegend 11-26c2a) 1:400, TCRβ-BV605 (BD H57-597) 1:200, TCRβ-FITC (ebioscience H57-597) 1:300, CD4-APC/Cy7 (BD GK1.5) 1:400, cKit-PE/Cy7 (biolegend 2B8) 1:300, cKit-PerCP-Cy5.5 (biolegend 2B8) 1:300, cKit-BV421 (biolegend 2B8) 1:300, Mac1-APC (ebioscience M1/70) 1:300, CD25-PE (biolegend PC61) 1:500, CD93-PE/Cy7 (ebioscience AA4.1) 1:300, NK1.1-APC (biolegend PK136) 1:300, AnnexinV-FITC (biolegend Lot: B206041) 1:1800, AnnexinV-eF450 (ebioscience; Lot: E11738-1633) 1:1000. FITC-F(ab’)₂ Fragment Goat Anti-Mouse IgM [1 μg/ml], μ Chain Specific (Jackson ImmunoResearch).

Schuler F., Weiss J.G., Lindner S.E., Lohmüller M., Herzog S., Spiegl S.F., Menke P., Geley S., Labi V, & Villunger A. (2017). Checkpoint kinase 1 is essential for normal B cell development and lymphomagenesis. Nature Communications, 8, 1697.

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