Flow cytometry antibodies

Manufactured by BioLegend

Sourced in United States

Flow cytometry antibodies are laboratory reagents used in flow cytometry analysis. They are designed to bind specific targets on cells or particles, enabling their identification and quantification through flow cytometric detection.

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Flow cytometry (10 citations) Antibodies for flow cytometry (10 citations) Flow antibodies (8 citations)

14 protocols using flow cytometry antibodies

Tumor Immune Cell Profiling by Flow Cytometry

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Tumours were excised, and orthotopic tumours were weighed. Tumours were chopped and incubated at 37°C in RPMI with collagenase IV (Sigma) and trypsin inhibitor (Life Technologies). Tumour pieces were further manually digested before being filtered through a 40 μM cell strainer, washed with PBS and centrifuged. The resulting pellet was resuspended in PBS with 2% FBS and stained with two separate panels of Flow cytometry antibodies. Samples were stained for 30 min at 4°C, washed with PBS and resuspended in 1% formalin before analysis on a Sony spectral flow cytometer (SP6800). Flow cytometry antibodies were purchased from BioLegend. Myeloid panel: CD11c FITC (no. 117305), CX3CR1 PE (no. 149006), Ly6G PE-Cy7 (no. 127617), SiglecF Brilliant Violet 421 (no. 155509), CD11b Pacific Blue (no. 101223), Ly6C Brilliant Violet 570 (no. 128030), I-A/I-E Brilliant Violet 510 (no. 107635), CD45 Brilliant Violet 711 (no. 103147), NK1.1 Brilliant Violet 785 (no. 108749), F4/80 APC (no. 123116).
Lymphoid panel: CD45 Brilliant Violet 711 (no. 103147), CD11b Pacific Blue (no. 101223), CD25 Alexa Fluor 488 (no. 102017), CD103 PE (no. 121405), PD-1 PE-Cy7 (no. 109109), CD4 Brilliant Violet 510 (no. 100553), B220 Brilliant Violet 605 (no. 103243), CD8α Brilliant Violet 785 (no. 100749), CD11c APC (no. 117309).

Stump C.T., Roehle K., Manjarrez Orduno N, & Dougan S.K. (2021). Radiation combines with immune checkpoint blockade to enhance T cell priming in a murine model of poorly immunogenic pancreatic cancer. Open Biology, 11(11), 210245.

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Corneal Cell Phenotyping by Flow Cytometry

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Transplanted corneas were collected and digested with collagenase and Dnase, and single-cell suspensions were prepared and filtered. Cells were incubated with an Fc receptor blocking antibody (R&D Systems, Minneapolis, MN, USA). After they were washed, the cells were incubated with flow cytometry antibodies from BioLegend (San Diego, CA, USA) or Thermo Fisher Scientific for 1 hour: PE-Cy7-anti-mouse CD45; Pacific Blue-anti-mouse CD11b Ab; FITC-anti-mouse CD4 Ab; APC-anti-mouse Ly-6G Ab; and PE-anti-mouse F4/80 Ab. Stained cells were rinsed and analyzed using an LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the results were analyzed using Summit 4.3 (Agilent Dako, Santa Clara, CA, USA). Dead cells were excluded by performing forward-scatter versus side-scatter analysis, and samples were subsequently analyzed by gating on CD45⁺ cells (Supplementary Fig. S1).

Wang S., Mittal S.K., Lee S., Herrera A.E., Krauthammer M., Elbasiony E., Blanco T., Alemi H., Nakagawa H., Chauhan S.K., Dana R, & Dohlman T.H. (2024). Effector T Cells Promote Fibrosis in Corneal Transplantation Failure. Investigative Ophthalmology & Visual Science, 65(1), 40.

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Multiparametric Analysis of γδ T Cells

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Single cell suspensions were stained with fixable viability dye (eBioscience) and antibodies for surface markers for 30 minutes at 4C. Next, cells were fixed with 4% formaldehyde for 10 minutes in the dark at room temperature. Flow cytometry antibodies were purchased from Biolegend (San Diego, CA), Invitrogen (Carlsbad, CA), BD Biosciences (Mountain View, CA) and eBioscience (San Diego, CA). Flow data were collected by LSR4 (BD Biosciences) and Attune Nxt (ThermoFisher). Data was analyzed with FlowJo (Tree Star, Ashland, OR). Unless otherwise indicated, γδ T cells are analyzed through lymphocyte size and viability gates, and γδTCR and Vγ3 positive cells are analyzed on a gated CD3+ population.

Wang J., Pajulas A., Fu Y., Adom D., Zhang W., Nelson A.S., Spandau D.F, & Kaplan M.H. (2022). γδ T cell-mediated wound healing is diminished by allergic skin inflammation. The Journal of investigative dermatology, 142(10), 2805-2816.e4.

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Profiling Immune Cell Subsets

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The different subsets of lymphocytes were identified using their specific markers. T cells were identified using CD3 (17A2); CD4 (GK1.5) and CD8 (53-6.7) markers were used to characterize the different subsets of T cells and macrophages were identified using F4/80 (BM8) and CD11b (M1/70) markers. Intracellular cytokine production (IL-6 [MP5-20F3], IFNγ [XMG1.2], and TNFα [MP6-XT22]) was determined in the CD4⁺ and CD8⁺ T cell subsets and F4/80⁺ and CD11b⁺ macrophages. Flow cytometry antibodies were purchased from BioLegend (San Diego, CA).

Manakkat Vijay G.K., Hu C., Peng J., Garcia-Martinez I., Hoque R., Verghis R.M., Ma Y., Mehal W.Z., Shawcross D.L, & Wen L. (2019). Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9. Cellular and Molecular Gastroenterology and Hepatology, 8(4), 609-623.

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Profiling Lung Immune Cells by Flow Cytometry

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IL-17A⁺ and IL-22⁺ CD4⁺ T-cells, γδ T-cells, NKT-cells and ILC3s in lung homogenates were determined based on surface marker expression (supplementary table S2) [25 (link)–27 (link)] using a BD FACSAriaIII. Flow cytometry antibodies were from Biolegend (Karrinyup, Australia) or BD Biosciences (North Ryde, Australia) (supplementary table S3, supplementary figure S1).

Starkey M.R., Plank M.W., Casolari P., Papi A., Pavlidis S., Guo Y., Cameron G.J., Haw T.J., Tam A., Obiedat M., Donovan C., Hansbro N.G., Nguyen D.H., Nair P.M., Kim R.Y., Horvat J.C., Kaiko G.E., Durum S.K., Wark P.A., Sin D.D., Caramori G., Adcock I.M., Foster P.S, & Hansbro P.M. (2019). IL-22 and its receptors are increased in human and experimental COPD and contribute to pathogenesis. The European respiratory journal, 54(1), 1800174.

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PBMC Cryopreservation and Flow Cytometry

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Commercially available cryopreserved PBMCs (AllCells) were thawed into DMEM with 10% FBS. Two million cells per condition (4 conditions) were spun down in Eppendorf tubes at 4°C for 5 min at 400 g, and resuspended in 100 μl PBS with 2% BSA. Each aliquot was incubated for 10 min with 10 μL of FcX block, followed by staining with flow cytometry antibodies (BioLegend) on ice for 30 min. Cells were washed three times with PBS with 2% BSA. Samples were then gated as described below and sorted directly into Buffer RLT (QIAGEN).

Hao Y., Hao S., Andersen-Nissen E., Mauck WM I.I.I., Zheng S., Butler A., Lee M.J., Wilk A.J., Darby C., Zager M., Hoffman P., Stoeckius M., Papalexi E., Mimitou E.P., Jain J., Srivastava A., Stuart T., Fleming L.M., Yeung B., Rogers A.J., McElrath J.M., Blish C.A., Gottardo R., Smibert P, & Satija R. (2021). Integrated analysis of multimodal single-cell data. Cell, 184(13), 3573-3587.e29.

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IM-CS-NPs/E7/ATP@ALG Nanovaccine Preparation

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Lactic acid-ethanolic acid (PLGA) copolymer 50/50 was obtained from Jinan Daigang Bioengineering Co; chitosan and PVA were purchased from Sigma; the Flow cytometry antibodies were provided by Biolegend, USA; LysoTracker Red were provided by Solarbio (L8010). DAPI Stain Solution (ab104139), Na⁺/K⁺-ATPase antibody (ab76020), HMGB1 antibody (ab79823), Donkey Anti-Rabbit IgG H&L antibody (ab150075) and Goat Anti-Rabbit IgG H&L antibody (ab150077) were purchased from Abcam; the antibodies for Calreticulin (27298–1-AP), HSP70 (10995–1-AP), HSP90 (13171–1-AP), HRP Goat Anti-Rabbit IgG antibody (SA00001-2) and ELISA kits (IL-1β, IL6, TNF-α and IFN-γ) were purchased from Proteintech (Scheme 1).Scheme 1

Schematic illustration for the preparation of IM-CS-NPs/E7/ATP@ALG nanovaccine and its effects inducing antitumor immunity

Liu Q., Hu Y., Zheng P., Yang Y., Fu Y., Yang Y., Duan B., Wang M., Li D., Li W., He J., Zheng X., Long Q, & Ma Y. (2023). Exploiting immunostimulatory mechanisms of immunogenic cell death to develop membrane-encapsulated nanoparticles as a potent tumor vaccine. Journal of Nanobiotechnology, 21, 326.

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Zirconium Oxychloride Octahydrate Synthesis

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Zirconium oxychloride octahydrate (ZrOCl₂·8H₂O) was purchased from Innochem (Beijing) Technology Co., Ltd. Singlet oxygen sensor green (SOSG) was purchased from Thermo Fisher Scientific. DAC was purchased from Bide Pharmatech Ltd. The culture medium and fetal bovine serum were purchased from Gibco Life Technologies. The Western blot antibody was purchased from Abcam. Flow cytometry antibodies were purchased from Biolegend Ltd. Various kinds of kits were purchased from Beyotime Institute of Biotechnology Biotechnology. Solvents are available from general reagent®, including N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and ethanol. Except as otherwise noted, all additional chemical reagents were bought from Sigma-Aldrich. The Animal Center of the Fourth Military Medical University (Xi'an, China) provided female BALB/c mice. In this study, animal protocols were reviewed and approved by the Fourth Military Medical University's Institutional Animal Care and Use Committee (approval number: 20190229).

Chen Z., Liu W., Yang Z., Luo Y., Qiao C., Xie A., Jia Q., Yang P., Wang Z, & Zhang R. (2023). Sonodynamic-immunomodulatory nanostimulators activate pyroptosis and remodel tumor microenvironment for enhanced tumor immunotherapy. Theranostics, 13(5), 1571-1583.

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Comprehensive Immune Cell Profiling

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Peripheral blood mononuclear cells or mononuclear cells isolated from liver tissue were stained with the following antibodies for phenotypic staining: CD3-APC/Cy7, CD8-BV510, CD8-Percp, CD11c-FITC, CD11c-PE, PD-1-APC, HLA-DR-BV421, CCR7-FITC, and CD45RA-BV510. After permeabilization using a Cytofix/Cytoperm Kit (BD Biosciences, Franklin Lakes, NJ, USA), cells were used for intracellular cytokine staining using an anti-GB-PE antibody. Anti-IL-2, anti-IFN-γ, and anti-TNF-α antibodies were used to stain intracellular cytokines after stimulation with ionomycin (1 μM) and phorbol-12-myristate-13-acetate (PMA) (100 ng/mL) for 6~8 h at 37°C in the presence of 5% CO₂. The anti-CD107a antibody was added at the same time as the stimulation. The flow cytometry antibodies were obtained from BioLegend (San Diego, CA, USA).

Gao L., Hong Z., Lei G., Guo A.L., Wang F.S., Jiao Y.M, & Fu J. (2023). Decreased granzyme-B expression in CD11c+CD8+ T cells associated with disease progression in patients with HBV-related hepatocellular carcinoma. Frontiers in Immunology, 14, 1107483.

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Immune Profiling Reagents Protocol

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Gemcitabine, Oxaliplatin, Pomalidomide and ZAedronic acid, Staphylococcus enterotoxin B (SEB) and WST-1 reagent were purchased from Sigma (Dorset, UK). Flow cytometry antibodies were purchased from Biolegend (London, UK) and R and D systems (Abingdon, UK). Cytomegalovirus, Epstein Barr Virus and Influenza virus (CEF) peptides were purchased from Sigma (Dorset, UK). CFSE reagent was purchased from Fisher Scientific—UK Ltd (Loughborough, UK).

Smith P.L., Yogaratnam Y., Samad M., Kasow S, & Dalgleish A.G. (2020). Effect of Gemcitabine based chemotherapy on the immunogenicity of pancreatic tumour cells and T-cells. Clinical & Translational Oncology, 23(1), 110-121.

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