Percp cy5.5 cd19

Manufactured by BioLegend

Sourced in United States

PerCP-Cy5.5-CD19 is a fluorochrome-conjugated antibody that binds to the CD19 surface protein, which is expressed on B cells. It can be used for the identification and enumeration of B cells in flow cytometry applications.

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5 protocols using percp cy5.5 cd19

Phenotypic Analysis of Freshly Isolated PBMCs

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The phenotype of freshly isolated PBMCs were analyzed by flow cytometry. The following fluorochrome-conjugated mAbs and corresponding isotype control antibodies were used: APC-cy7-CD3 (clone: UCHT1; Biolegend), Percp-cy5.5-CD14 (clone: M5E2; Biolegend), Percp-cy5.5-CD19 (clone: HIB19; Biolegend), APC-CD16 (clone: 3G8; Biolegend), PE-cy7-CD56 (clone: HCD56; Biolegend), PE-CD160 (clone: BY55; Biolegend), PE-IgM (clone: MM-30; Biolegend), Brilliant Violet 421-CD69 (clone: FN50; Biolegend), Brilliant Violet 421-IgG1 (clone: MOPC-21; Biolegend), Alexa Fluor^® 488-GLUT1 (clone: 202915; R&D Systems), PE-CD36 (clone: CB38; BD PharMingen™), PE-IgM (clone: G155-228; BD PharMingen™), APC-CD71 (clone: CY1G4; Biolegend), APC-IgG2a (clone: MOPC-173; Biolegend), Brilliant Violet 510-CD98 (clone: UM7F8; BD OptiBuil™), and Brilliant Violet 510-IgG1 (clone: X40; BD OptiBuil™). Fixable Viability Stain 620 (BD Horizon™, USA) was used to evaluate cell viability in all experiments. The phenotypic analysis above was conducted using a BD FACS Canto II Flow Cytometer and analyzed by FlowJo 10.4 software (Ashland, OR, USA).

Sun Z., Li Y., Zhang Z., Fu Y., Han X., Hu Q., Ding H., Shang H, & Jiang Y. (2022). CD160 Promotes NK Cell Functions by Upregulating Glucose Metabolism and Negatively Correlates With HIV Disease Progression. Frontiers in Immunology, 13, 854432.

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Multicolor Flow Cytometry Panel

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AF488: CD45 (clone: 30-F11, BioLegend); PerCp/Cy5.5: CD19 (clone: 6D5, BioLegend); APC: F4/80 (clone: BM8, BioLegend); APC-Cy7: CD4 (clone: GK1.5, BioLegend); PE-Cy7: CD8a (clone: 53-6.7, BioLegend); PE: CD49b (clone: DX5, BioLegend); BV650: CD3 (clone: 17A2, BioLegend); BV421: Ly6G (clone: 1A8, BioLegend).

Gong H.H., Worley M.J., Carver K.A., Goldstein D.R, & Deng J.C. (2023). Neutrophils drive pulmonary vascular leakage in MHV-1 infection of susceptible A/J mice. Frontiers in Immunology, 13, 1089064.

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Multiparametric Flow Cytometry for T-Cell Analysis

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Allophycocyanin-conjugated (APC) mAbs to Foxp3 and CD62L; Phycoerythrin-conjugated (PE) mAbs to CD4, CD44, TNF-α, and LAP; PerCPCy5.5-conjugated mAbs anti-CD4, IFN-γ, and rat IgG2a isotype control from BD Biosciences (San Jose, CA, United States) were used as markers for T cells. PerCP-Cy5.5-CD19, FITC-CD25, and PE-conjugated anti IL-10 from Biolegend (San Diego, CA, United States). For surface antigen detection, cells were labeled with monoclonal antibodies for 30 min at 4°C. For intracellular labeling of cytokines and transcription factors, a fixation/permeabilization kit (e-Bioscience, San Diego, CA, United States) was used after this step. Samples were then incubated for 30 min with a solution containing the appropriate antibodies. After washing with PBS containing 0.5% FBS, samples were fixed with 3% paraformaldehyde for 30 min, washed and stored in PBS at 4°C. Cells were acquired using a FACSCanto II cytometer (Becton Dickinson, East Rutherford, NJ, United States) and data was analyzed by FlowJo software (Tree Star, Ashland, OR, United States). At least 30,000 events were acquired for each analysis. Gating strategies are detailed in Supplementary Material (Supplementary Figure S1).

Gusmao-Silva G., Aguiar S.L., Miranda M.C., Guimarães M.A., Alves J.L., Vieira A.T., Cara D.C., Miyoshi A., Azevedo V.A., Oliveira R.P, & Faria A.M. (2020). Hsp65-Producing Lactococcocus lactis Prevents Antigen-Induced Arthritis in Mice. Frontiers in Immunology, 11, 562905.

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Splenocyte Activation and Intracellular Cytokine Staining

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Splenocytes (5 × 10⁶/mL) were stimulated with plate-bound anti-CD3 antibodies (2 μg/mL) and soluble anti-CD28 antibodies (2 μg/mL) for 3 days, and brefeldin-A (5 μg/mL: Biolegend) for the final 16 h of culture. Cells were resuspended in flow buffer (PBS with 0.1% sodium azide and 1% FBS), and stained with anti-CD16/32 TruStain fcX (1: 200, Biolegend) and a live/dead viability dye (BV-510 at 1:500; BD Biosciences) for 30 min at 4 °C. Staining of surface markers (APC-CD4, FITC-CD8α, PercpCy5.5-CD19, all Biolegend) was performed for 30 min at 4 °C in the dark. Cells were fixed, permeabilized using the FIX/PERM set (Biolegend) and blocked in 5% rat serum for 10 min at room temperature prior to intracellular staining for IL-4 with PE-Cy7-anti-IL-4 (Biolegend) for 20 min at room temperature. Data was analyzed using BD FACSDIVA software (BD Biosciences, San Jose, CA, USA).

Matisz C.E., Faz-López B., Thomson E., Al Rajabi A., Lopes F., Terrazas L.I., Wang A., Sharkey K.A, & McKay D.M. (2017). Suppression of colitis by adoptive transfer of helminth antigen-treated dendritic cells requires interleukin-4 receptor-α signaling. Scientific Reports, 7, 40631.

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Lificiguat and Immune Cell Analysis

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Lificiguat (YC-1) was obtained from Selleck (United States) (Cat: S7958). CoCl₂ · 6H₂O was purchased from Merck (Germany) (Cat: C8661). The following fluorescence-labeled antibodies were purchased from BD (United States), R&D (United States), or Biolegend (United States): Percp-cy5.5-CD4 (GK1.5), APC-PD-1 (29F.1A12), FITC-CD3 (145-2C11), Percp-cy5.5-CD19 (6D6), APC-cy7-CD8 (54-6.7), PE-cy7-CD11b(M1/70), APC-CD62L (MEL-14), PE-CD25(BC96), PE-CD40L (MR1), APC-CD69 (H12F3), APC-IL-17(TC11-18H10.1), BV421-CXCR5 (L138D7), APC-IFN-γ (XMG1.2), PE-IL10 (JES5-16E3), PE-IL4 (11B11), PE-IL2 (JES6-5H4), anti-mouse CD16/CD32 (2.4G2), Alexa Fluor 488-NFATc1(7A6), APC- HIF-1α(241812). Anti-NFATc1 rabbit mAb (D15F1), anti-tubulin antibody (Cat: 2144S), anti-histone H3 rabbit mAb (D1H2), and HRP-labled secondary antibodies (Cat: L3012-2) were purchased from cell signaling technology (CST, United States). The eFluor506-FVD (Cat: 65-0863-14) was purchased from eBioscience (United States).

Wei H., Xie A., Li J., Fang C., Liu L., Xing J., Shi F., Mo F., Chen D., Xie H., Yang Q., Pan X., Tang X, & Huang J. (2022). PD-1+ CD4 T cell immune response is mediated by HIF-1α/NFATc1 pathway after P. yoelii infection. Frontiers in Immunology, 13, 942862.

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