Tnfα apc cy7

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The TNFα-APC-Cy7 is a reagent used in flow cytometry analysis. It is a fluorescent-labeled antibody that binds to the tumor necrosis factor alpha (TNFα) molecule, which is a cytokine involved in inflammatory responses. The APC-Cy7 fluorochrome is used to label the antibody, allowing for the detection and quantification of TNFα-expressing cells through flow cytometry.

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APC-Cy7 (76 citations)

4 protocols using tnfα apc cy7

Multiparameter Flow Cytometry of Immune Cells

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CD45.2-V500 and TNFα-APC-Cy7 were purchased from BD Biosciences (San Jose, California). KLRG1-FITC, Eomes-PE, CD107a-PE, CD107b-PE, CD27-PE, CD127-PE-Cy5, CD127-PerCP-Cy5.5, Tbet-PerCP-Cy5.5, IFNγ-PerCP-Cy5.5, Eomes-PErCP-efluor710, CD45.1-PECy7, KLRG1-PE-Cy7, Tbet-PE-Cy7, IRF4-AlexaFluor647, CD44-AlexaFluor700, CD62L-APC-eFluor780, CD44-eFluor450, KLRG1-eFluor450, IFNγ-eFluor450, CD90.2-APC-eFluor780, CD45.1-APC-eFluor780, IL-2-PerCP-Cy5.5 were purchased from eBioscience (San Deigo, California). CD8-PE-TexasRed, GranzymeB-PE, GranzymeB-APC, Live-Dead-Violet, Live-Dead-Aqua and goat-anti-rabbit IgG-AlexaFluor647 and -AlexaFluor488 were purchased from Life Technologies (Grand Island, New York). H2D^b-GP33 monomers were prepared at UMMS; LCMV-specific (H2D^b-NP396, H2D^b-GP276) and Influenza A PR8-OVA_I-specific (H2K^b-OVA257) monomers were obtained from the NIH Tetramer Core Facility (Atlanta, Georgia). Intracellular TCF1 staining was performed using rabbit-anti-mouse TCF1 (Cell Signaling Technology, Danvers, Massachusetts) followed by staining with goat-anti-rabbit secondary (Life Technologies). Samples were analyzed on an LSRII flow cytometer (Becton Dickinson), and data were analyzed using FlowJo (Tree Star).

Nayar R., Schutten E., Bautista B., Daniels K., Prince A.L., Enos M., Brehm M.A., Swain S.L., Welsh R.M, & Berg L.J. (2014). Graded levels of IRF4 regulate CD8+ T cell differentiation and expansion, but not attrition, in response to acute virus infection. Journal of immunology (Baltimore, Md. : 1950), 192(12), 5881-5893.

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Multiparameter Flow Cytometry Analysis

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Fluorochrome-conjugated anti-mouse antibodies (anti-CD45.2-PE-Cy7, anti-CD3-APC-Cy7, anti-CD4-APC, anti-CD8-PerCP-Cy5.5, NK1.1-PE, CD11b-APC, Ly6G-PE, Ly6C-FITC, IFNγ APC, TNFα APC-Cy7, anti-CXCR3-PE, Streptavidin-APC and 7-AAD) were purchased from BD PharMingen and eBioscience. AnnexinV FITC was purchased from Biolegend. Trp-2 peptide (Trp2180: SVYDFFVWL) was purchased from Peptide 2.O Inc. Anti-m-OX-40 agonistic Ab (Clone OX-86) and Anti-m-PD-1 antagonistic Ab (Clone: RMP1-14) were purchased from BioXcell. Anti-mouse BLT1 antibody conjugated to biotin was developed in the lab (unpublished data).

Chheda Z.S., Sharma R.K., Jala V.R., Luster A.D, & Haribabu B. (2016). Chemoattractant receptors BLT1 and CXCR3 regulate antitumor immunity by facilitating CD8+ T cell migration into tumors. Journal of immunology (Baltimore, Md. : 1950), 197(5), 2016-2026.

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Splenocyte Cytokine Profiling of Macrophages

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Splenocytes were cultured, brefeldin A (10 μg ml⁻¹) was added after 2 h, and cells were incubated further for another 3 h. Following fixation and permeabilization with 2% formaldehyde and 0.5% saponin (Rectapur), intracellular cytokine staining was performed using F4/80-APC, CD11b-PE-Cy7, IL1-APC-Cy7, IL10-APC-Cy7, TNF-α-APC-Cy7 and IL-4-APC-Cy7 (BD biosciences, CA USA). The frequency of IL-1, IL-10, TNF-α or IL-4-positive cells were determined within the F4/80^highCD11b^high population for splenic macrophages.

Gaharwar U.S., Kumar S, & Rajamani P. (2020). Iron oxide nanoparticle-induced hematopoietic and immunological response in rats. RSC Advances, 10(59), 35753-35764.

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Tumor Immune Profiling by Flow Cytometry

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Tumor xenografts were cut into pieces and digested with collagenase IV (Sigma, USA) and DNAse I (Sigma, USA) for 30 min at 37°C. Then, cell suspensions were passed through a 70-μm strainer to remove undigested tissues. Erythrocytes were removed by Red Blood Cell Lysis Solution (Qiagen, USA). For cell surface staining, 1 × 10⁶ cells were incubated with anti-Fc receptor blocking antibody at 4°C for 30 min. For intracellular staining, 1 × 10⁶ cells were fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BD Bioscience, USA) according to the manufacturer’s instructions. Then, cells were stained with the indicated antibodies for 30 min at 4°C. Flow cytometry was performed using a BD Influx cell sorter (BD Bioscience). Flow cytometry data were analyzed by FlowJo version 10 (FlowJo, USA). The flow cytometry antibodies used in our study were CD45 APC, CD4 PE, CD8a V450, CD25 APC-CY7, FoxP3 PE-CY7, IFN-γ PE-CY7, IL-4 APC-CY7, Gr-1 V450, Ly6C FITC, CD11 b PE, F4/80 APC-CY7, Tim-3 PE-CY7, PD-1 PerCP CY5.5, T-bet FITC, GATA3 APC, TNF-α APC-CY7, anti-Ki67 FITC, and anti-Granzyme B PE, all from BD Bioscience.

Wang D., Zou F., Li Y., Hu J, & Gao L. (2024). Targeting MELK improves PD-1 blockade efficiency in cervical cancer via enhancing antitumor immunity. Molecular Therapy Oncology, 32(1), 200759.

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