Percp cy5.5 anti cd19

Manufactured by BioLegend

Sourced in United States

PerCP-Cy5.5 anti-CD19 is a fluorescently labeled antibody that binds to the CD19 antigen, which is expressed on the surface of B cells. It is designed for use in flow cytometry applications to identify and quantify B cell populations.

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5 protocols using percp cy5.5 anti cd19

Multiparametric Flow Cytometry Profiling

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For ABCs detecting, PBMCs/B cells to be detected were incubation with FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4 °C, then stained with BV421 anti-CD11c (BioLegend) and percp-Cy5.5 anti-CD19 (BioLegend) at 4 °C for 30 minutes. For intracellular staining, cells were surface stained, fixed using eBio fix/perm kit (eBioscience), washed and stained with PE-Cyanine7 anti-T-bet (BioLegend) in 1 × eBio fix/perm buffer for 1 hour at 4 °C. Mouse IgG1 kappa Isotype Control, PE-Cyanine7 (eBioscience) was used as isotype control for T-bet staining.
For RNASE2 detecting, PBMCs to be detected were incubation with FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4 °C, then stained with APC anti-CD14 and viability dye efour 506 (Invitrogen) at 4 °C for 30 minutes. For intracellular staining, cells were surface stained, fixed with fixation/permeabilization solution (Cytofix/Cytoperm kit, BD), washed and stained with RNASE2 antibody (Invitrogen) in 1× Perm/Wash buffer for 1 hour at 4 °C, then stained with AlexaFlour 488 Goat anti-rabbit IgG (FCMACS). Cells stained with CD14 and AlexaFlour 488 Goat anti-rabbit IgG were used as isotype control for RNASE2 staining.
Then Cells were analyzed on a BD FACSAria III flow cytometer (BD Biosciences) using FACSDiva software. Cells were analyzed on a BD FACSAria III flow cytometer (BD Biosciences) using Flowjo VX software.

Zhu Y., Tang X., Xu Y., Wu S., Liu W., Geng L., Ma X., Tsao B.P., Feng X, & Sun L. (2022). RNASE2 Mediates Age-Associated B Cell Expansion Through Monocyte Derived IL-10 in Patients With Systemic Lupus Erythematosus. Frontiers in Immunology, 13, 752189.

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Multiparametric Flow Cytometry Analysis

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1–2 × 10⁶ cells were placed in 200 uL of PBS1X with 2% FBS on ice and antibodies were added, mixed, and incubated for 30 min. Then, cells were washed with PBS1X and centrifuged at 800×g for 5 min. Finally, samples were acquired in a CytoFLEX LX cytometer (Beckman Coulter). Analysis was performed using CytExpert software. The following anti human antibodies were used: AF488 anti-GRP78 (Clone: C38), AF700 anti-CD45 (Clone: HI30), APC anti-CD34 (Clone: 561), PE/Cy7 anti-CD38 (Clone: HB-7), PerCP/Cy5.5 anti-CD19 (Clone: HIN19), PE/Dazzle anti-CD10 (Clone: HI10a), Brilliant Violet 421 anti-CD184 (clone: 12G5) and the Zombie Aqua™ Fixable Viability Kit was used according to the manufacture (Biolegend).

Angeles-Floriano T., Rivera-Torruco G., García-Maldonado P., Juárez E., Gonzalez Y., Parra-Ortega I., Vilchis-Ordoñez A., Lopez-Martinez B., Arriaga-Pizano L., Orozco-Ruíz D., Torres-Nava J.R., Licona-Limón P., López-Sosa F., Bremer A., Alvarez-Arellano L, & Valle-Rios R. (2022). Cell surface expression of GRP78 and CXCR4 is associated with childhood high-risk acute lymphoblastic leukemia at diagnostics. Scientific Reports, 12, 2322.

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Multiparameter Flow Cytometric Analysis

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The cells were blocked with purified anti-mouse CD16/32 and stained with the following fluorescent conjugated antibodies for 30 minutes on ice: PerCP-Cy5.5 anti-CD19, APC anti-CD23, and Brilliant Violet 785 anti-MHC II from BioLegend, US. Invitrogen^™ DAPI (4’,6-Diamidino-2-Phenylindole, Dihydrochloride) (#D1306) was used for staining the dead cells. After the staining, the samples were run on the Flow cytometer machine Becton Dickinson LSRFortessa, USA or Cytek Biosciences Cytek Aurora, USA. Data analysis and quantification were performed using FlowJo v10.6.2.

van der Vlugt-Bergmans C.J., Meeuwsen P.J., Voragen A.G, & van Ooyen A.J. (2000). Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme. Applied and Environmental Microbiology, 66(1), 36-41.

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Germinal Center B Cell Identification

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After live/dead staining using the Live/dead Fixable Dead Cell Stain kit (Thermo Fisher Scientific), and surface staining with Percp/cy5.5-anti-CD19, FITC-anti-CD95, and APC-anti-GL-7, or APC-anti-CD11c (Biolegend), the cells were washed and fixed in 1% paraformaldehyde solution. The samples were run through an LSRII/Fortessa (Mountain View, CA), and data were analyzed using FlowJo software. Single live CD19⁺ cells were gated firstly for analysis of germinal center B cells (Supplementary Figure 3C).

Yang W., Xiao Y., Huang X., Chen F., Sun M., Bilotta A.J., Xu L., Lu Y., Yao S., Zhao Q., Liu Z, & Cong Y. (2019). Microbiota metabolite short-chain fatty acids facilitate mucosal adjuvant activity of cholera toxin through GPR43. Journal of immunology (Baltimore, Md. : 1950), 203(1), 282-292.

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Flow Cytometry Antibody Panel for Immune Cell Analysis

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Antibodies used for flow cytometry include the following: FITC–anti-CD19 (rat IgG2a; 1:200, #115506; BioLegend), PE–anti-CD45.1 (mouse [A.SW] IgG2a; 1:100, #110707; BioLegend), PE/Cy7–anti-Thy1.1 (mouse IgG1; 1:100, #202518; BioLegend), BV421–anti-CD4 (rat IgG2b; 1:150, #100438; BioLegend), APC–anti-CD45.2 (mouse [SJL] IgG2a; 1:100, #109813; BioLegend), V500–anti-CD3e (Syrian hamster IgG2; 1:50, #560771; BD Biosciences), FITC–anti-CD25 (rat IgG2b; 1:100, #101907; BioLegend), PerCP/Cy5.5–anti-CD19 (rat IgG2a; 1:100, #152405, BioLegend), PerCP/Cy5.5–anti-NK1.1 (mouse IgG2a; 1:100, #108727; BioLegend), PerCP/Cy5.5–anti-CD8b (rat IgG2b; 1:100, #126609; BioLegend), BV605–anti-CD44 (rat IgG2b; 1:100, #103047; BioLegend), FITC–anti-CD8a (rat IgG2a; 1:100, #100705; BioLegend), BV510–anti-CD11b (rat IgG2b; 1:100, #101245; BioLegend), BV421–anti-Ly6C (RçAT IgM; 1:100, #562727; BD Biosciences), and PerCP/Cy5.5–anti-Ly6G (rat IgG2a; 1:100, #127615; BioLegend). For mass cytometry experiments, metal-tagged antibodies were obtained from Fluidigm or conjugated using the Maxpar X8 Antibody Labeling kit according to the manufacturer’s instructions (Fluidigm). Clone and tag information can be found in Table S1.

Warner J.D., Irizarry-Caro R.A., Bennion B.G., Ai T.L., Smith A.M., Miner C.A., Sakai T., Gonugunta V.K., Wu J., Platt D.J., Yan N, & Miner J.J. (2017). STING-associated vasculopathy develops independently of IRF3 in mice. The Journal of Experimental Medicine, 214(11), 3279-3292.

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