Tnf α pe

Manufactured by BD

Sourced in United States

TNF-α-PE is a laboratory reagent that enables the detection and quantification of tumor necrosis factor alpha (TNF-α) in biological samples using flow cytometry. It consists of a fluorescent dye, phycoerythrin (PE), conjugated to an antibody specific for TNF-α.

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Anti-mouse TNF-α-PE PE-Cy7-conjugated TNF-α mAbs Anti–TNF-α–PE (MP6-XT22; TNF-α-PE-Cy7 Anti-human PE TNF-α PE‐labelled rat anti‐mouse TNF‐α antibody Anti-TNF-α-PE PE-Cy7-conjugated anti-TNF-α TNF-α

22 protocols using tnf α pe

Intracellular Cytokine Staining of T Cells

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For intracellular cytokine staining, T cells were rested with IL-2 (100 U/mL) overnight and then stimulated with PepMixes encompassing viral or control proteins (JPT) at 0.5 nmol/μL, along with anti-CD49d/CD28 antibodies (BD Biosciences) at 1:1,000 for co-stimulation and Golgi stop at 1 μL/mL. SEB and Actin were utilized as controls. T cells were stained with cell surface markers (CD3, CD4, and CD8), followed by permeabilization with Cytofix/Cytoperm (BD Biosciences), washing, and staining with IFN-γ-APC (BioLegend), IL-2-fluorescein isothiocyanate (FITC), and TNF-α-PE (BD Biosciences).

Dave H., Luo M., Blaney J.W., Patel S., Barese C., Cruz C.R., Shpall E.J., Bollard C.M, & Hanley P.J. (2017). Toward a Rapid Production of Multivirus-Specific T Cells Targeting BKV, Adenovirus, CMV, and EBV from Umbilical Cord Blood. Molecular Therapy. Methods & Clinical Development, 5, 13-21.

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Multiparametric T cell Characterization

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T cells were washed in staining buffer (SB) consisting of phosphate-buffered saline (PBS) containing 0.1% human serum albumin (HSA) and 0.1% sodium azide before staining for 20 min at RT. The cells were then washed in SB and fixed in SB containing 1% paraformaldehyd. For intracellular staining, T cells were stimulated for 6 h or overnight with APCs, loaded or not with p573, at a T-cell to target ratio of 1:2 and in the presence of BD GolgiPlug and BD Golgistop at a 1/1,000 dilution. Cells were stained both extracellular and intracellular using the PerFix-nc kit according to the manufacturer's instructions (Beckman Coulter Inc., USA). The following antibodies were used: Vβ3- FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor450, CD4-eFluor 450, CD4-PE-Cy7, CD8-APC, CD8-eFluor 450, CD8-PE-Cy7, CD56-PE-Cy5.5 (BD Biosciences, USA) and CD107a-PE-Cy5 (BD Biosciences, USA), CXCR2-PE, IFNγ-FITC, IL-2-APC, TNF-α-PE (BD Biosciences, USA), CD261/TRAIL-R4-PE (BD Biosciences, USA). MART-1 (aa 26–35)-specific TCR was detected with dextramer staining (Immudex) following the manufacturer's recommendations. All antibodies were purchased from eBioscience, except where noted. Cells were acquired on a BD LSR II flow cytometer and the data analyzed using FlowJo software (Treestar Inc.).

Inderberg E.M., Wälchli S., Myhre M.R., Trachsel S., Almåsbak H., Kvalheim G, & Gaudernack G. (2017). T cell therapy targeting a public neoantigen in microsatellite instable colon cancer reduces in vivo tumor growth. Oncoimmunology, 6(4), e1302631.

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T-cell Transduction and Cytokine Assay

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T-cell transduction, cytokine staining and bioluminescence (BLI)-based killing assay have been described in details elsewhere [9 (link)]. Briefly, primary T-cell transduction with retroviral particles was performed by double spinoculation followed by expansion on CD3/CD28 beads. The staining of the CAR was performed with Biotin-SP-AffiniPure F (ab′)2 Fragment Goat Anti-Mouse IgG (Jackson ImmuResearch, USA), followed by a secondary staining with Streptavidin`-PE (Biolegend, USA). CAR-expressing T cells were co-cultured with cells positive or negative for the target antigen, and TNF-α was detected by flow cytometry using TNF-α-PE (MAb11, BD Biosciences). The samples were run on a BD FACSCanto flow cytometer (BD Biosciences, USA), and data were analyzed using FlowJo software (Treestar Inc., USA). BLI killing assay was also based on a co-culture experiment where target cells permanently expressed firefly Luciferase gene, and killing was correlated to the luminescence signal [10 (link)].

Köksal H., Baken E., Warren D.J., Løset G.Å., Inderberg E.M, & Wälchli S. (2019). Chimeric antigen receptor preparation from hybridoma to T-cell expression. Antibody Therapeutics, 2(2), 56-63.

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Phenotypic and Functional Analysis of Antigen-Specific T Cells

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To analyze the magnitude and phenotype of the HIV-1-, FLAG- or VACV-specific T cell immune responses, 2 × 10⁶ splenocytes (erythrocyte-depleted) seeded on 96-well plates were stimulated for 6 h in complete Roswell Park Memorial Institute (RPMI) 1640 medium (100 units/mL of penicillin/100 μg/mL of streptomycin, 2 mM L-glutamine, 10 mM Hepes and 0.01 mM β-mercaptoethanol) with 10% FCS, anti-CD107a-FITC (BD Biosciences), 1 µL/mL Golgiplug (BD Biosciences), monensin 1X (Invitrogen) and 5 µg/mL of the different HIV-1 clade B consensus peptide pools or 5 µg/mL of FLAG peptide or 10 µg/mL of VACV E3 peptide. After stimulation, splenocytes from immunized mice were stained for surface markers, fixed/permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and stained intracellularly with the following fluorochrome-conjugated antibodies: IL-2-APC, IFN-γ-PeCy7 and TNF-α-PE for functional analyses and CD3-PECF594, CD4-APCCy7, CD8-V500, CD127-PerCPCy5.5 and CD62L-Alexa700 for phenotypic analyses (all from BD Biosciences). The dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen).

Perdiguero B., Sánchez-Corzo C., S. Sorzano C.O., Mediavilla P., Saiz L., Esteban M, & Gómez C.E. (2019). Induction of Broad and Polyfunctional HIV-1-Specific T Cell Responses by the Multiepitopic Protein TMEP-B Vectored by MVA Virus. Vaccines, 7(3), 57.

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Cytokine Production in Stimulated T Cells

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Mononuclear cells from blood were isolated and viably cryopreserved. Cells were thawed, rested overnight, and 2×10⁶ cells were stimulated with 2μg/ml of cognate peptides for 6h in RPMI containing 10% human serum in the presence of 5 μg/ml of Brefeldin A (Sigma-Aldrich). Non-stimulated cells, as well as cells stimulated with super antigen SEM (SEA (staphylococcal enterotoxin A) + SEB (staphylococcal enterotoxin B) (Sigma-Aldrich), were used as controls. These cells were then stained with the following surface markers for 15 min at 4°C: CD4 (clone L200-PerCP-Cy5.5, BD Biosciences), CD8 (CD8–PE–Texas Red (ECD), Cedarlane), CD95 (clone DX2-PE-Cy5, BD Biosciences), CD28 (clone CD28.2-Pacific Blue, custom made, BD Biosciences), CCR7 (clone 3D12-PE-Cy7, BD Biosciences), PD-1 (clone MIH4-FITC, BD Biosciences). Cells were fixed for 10 minutes in 100 μl 2% paraformaldehyde at room temperature (25°C). To stain cells with antibodies specific for intracellular cytokines (IFN-γ-Alexa-700, IL-2-APC, TNF-α-PE; BD Biosciences), we incubated the cells with antibodies in 0.25% saponin (Sigma-Aldrich) for 30 minutes at 25°C and analyzed them using the BD LSRII flow cytometer. Between 250,000 and 1×10⁶ events were acquired for each condition. Data were then analyzed using DIVA software (BD Biosciences) and the frequency of cytokine producing T cells is presented.

Gordon S.N., Liyanage N.P., Doster M.N., Vaccari M., Vargas-Inchaustegui D.A., Pegu P., Schifanella L., Shen X., Tomaras G.D., Rao M., Billings E.A., Schwartz J., Prado I., Bobb K., Zhang W., Montefiori D.C., Foulds K.E., Ferrari G., Robert-Guroff M., Roederer M., Phan T.B., Forthal D.N., Stablein D.M., Phogat S., Venzon D.J., Fouts T, & Franchini G. (2016). Boosting of ALVAC-SIV Vaccine-Primed Macaques with the CD4-SIVgp120 Fusion Protein Elicits Antibodies to V2 Associated with a Decreased Risk of SIVmac251 Acquisition. The Journal of Immunology Author Choice, 197(7), 2726-2737.

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Flow Cytometry Antibody Staining Protocol

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The following antibodies were used for culturing or surface and intracellular cytokine staining (ICS) for flow cytometry: CD28 (CD28.2, BD), CD49d (9F10, BD), CD3-PB (SP34-2, BD), CD4-BV510 (L200, BD), CD8-PB (RPA-T8, BD), IFN-γ-APC (4S.B3, BD), IFN-γ Brilliant Violet 711 (4S.B3, Biolegend), TNF-α-PE (MAb11, BD), TNF-α-PE-Cy7 (MAb11, BD), IL-17-PE (eBio64CAP17, eBioscience), IL-22-biotinylated (anti-human IL-22, RD), Streptavidin-Pacific blue (Invitrogen), Perforin-biotinylated (Pf-344, Mabtech), Caspase 3-AF647 (C92-605,BD), and anti-Vγ2-FITC (7A5, Pierce).
After staining, the cells were fixed and subjected to flow cytometry. Lymphocytes were gated based on forward- and side-scatters, and at least 40,000 gated events were analyzed using Summit Data Acquisition and Analysis Software (DakoCytomation).

Yang E., Yang R., Guo M., Huang D., Wang W., Zhang Z., Chen C., Wang F., Ho W., Shen L., Xiao H., Chen Z.W, & Shen H. (2018). Multidrug-resistant tuberculosis (MDR-TB) strain infection in macaques results in high bacilli burdens in airways, driving broad innate/adaptive immune responses. Emerging Microbes & Infections, 7, 207.

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Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes

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Flow cytometry analysis was performed by using a BD FACSVerse (BD Biosciences, Milan, Italy) and the following monoclonal anti-human antibodies: CD45-APC-H7, CD3ɛ-PerCP, CD3ɛ-FITC, CD8α-V500, CD56-V450, CD56-PE-Cy7, CD19-FITC, IFN-γ-FITC, IFN-γ-PE, IL-17A-V450, IL-17F-PE, TNFα-PE (all from BD Pharmingen, Milan, Italy), IL-21-PE, IL-22-APC, IL-6-PerCP, T-bet-PE-Cy7 (clone 4B10), RORγt-APC (clone AFKJS-9), RORγt-PE (clone AFKJS-9) (all from eBioscience) and CD68 (BioLegend, San Diego, CA, USA). TILs isolated from mouse colon tumors were stained with the following antibodies: CD3ɛ-Pacific Blue, CD8α-FITC, CD45-APC-Cy7, CD49b-PE (clone DX5), CD19-APC (all from BD Pharmingen) and F4/80-Pacific Blue (BioLegend). In parallel, cells were stained with the respective control isotype antibodies.

De Simone V., Franzè E., Ronchetti G., Colantoni A., Fantini M.C., Di Fusco D., Sica G.S., Sileri P., MacDonald T.T., Pallone F., Monteleone G, & Stolfi C. (2014). Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth. Oncogene, 34(27), 3493-3503.

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Multiparameter Flow Cytometry Panels

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The following Abs were used for culture or surface and intracellular cytokine staining for flow cytometry: CD28 (CD28.2, BD), CD49d (9F10, BD), CD3-PECY7 (SP34-2, BD), CD3-PB (SP34-2, BD), CD4-APC (L200, BD, used for measuring CD4 expression after CD4 depleting Ab treatment), CD8-PB (RPA-T8, BD), CD8-APC (RPA-T8, BD), CD8-PE (RPA-T8, BD), IFN-γ-Allophycocyanin (4S.B3, BD), IFN-γ-PE (4S.B3, BD), TNF-α-PE (MAb11, BD), TNF-α-APC (MAb11, BD), TNF-α-PB (MAb11, eBioscience), IL-17-PE (eBio64CAP17, eBioscience), IL-17-Alexa647 (eBio64CAP17, eBioscience), IL-22-biotinylated (anti-human IL-22, RD, see reference(44 (link))), Streptavidin-Pacific blue (invitrogen). IL-4-APC (8D4-8, eBioscience), Perforin-biotinylated (Pf-344, Mabtech)

Yao S., Huang D., Chen C.Y., Halliday L., Wang R.C, & Chen Z.W. (2014). CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes. Journal of immunology (Baltimore, Md. : 1950), 192(5), 2120-2132.

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Intracellular Cytokine Production in T and NKT Cells

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To determine whether GCR and SIRT1 are associated with intracellular cytokine production in CD8+ and CD8– T and NKT-like cells, aliquots of blood were stimulated and treated as described above. Following washing of permeabilized cells, appropriately diluted GCR antibody (MCA2469, Bio-Rad, Sydney, Australia) was added to cells for 15 min. Cells were further washed and stained with anti-mouse IgG1 V450 secondary antibody for 15 min. Cells were then washed and stained with SIRT1 Alexa-Fluor 488, IFNγ PE (BD), TNFα PE (BD), CD3 perCP.CY5.5 (BD, Sydney, Australia), CD28 PECY7 (BD), CD56 APC (Beckman Coulter, Sydney, Australia), CD8 APC.CY7 (BD), and CD45 V500 (BD) for 15 min in the dark at room temperature. Appropriate controls and cells were analyzed as above.

Hodge G., Tran H.B., Reynolds P.N., Jersmann H, & Hodge S. (2020). Lymphocyte senescence in COPD is associated with decreased sirtuin 1 expression in steroid resistant pro-inflammatory lymphocytes. Therapeutic Advances in Respiratory Disease, 14, 1753466620905280.

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Cytokine and Surface Marker Analysis

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Cytokines and surface markers were assessed by flow cytometry with a FACS Canto II (BD Biosciences). After sorting CD4⁺T cells, First, we used phorbol-12-myristate-13-acetate (PMA) and ionomycin with the addition of GolgiPlug (BD Biosciences) to stimulate and activate the cells for 4 h for cytokines detection, including IFN-γ, IL-6, and TNF-α. Surface marker staining was done at 4°C in the dark for 30 min. The following antibodies were used for flow cytometry: anti-human CD4-FITC (clone: RPA-T4), IFN-γ-PercpCy5.5 (clone: B27), IL-6-PE (clone: MQ2-13A5), and TNF-α-PE (clone: MAb11) were purchased from BD Biosciences. Events were collected and analyzed with FlowJo software (Tree Star, Inc.).

Tan L., Zhao M., Wu H., Zhang Y., Tong X., Gao L., Zhou L., Lu Q, & Zeng J. (2021). Downregulated Serum Exosomal miR-451a Expression Correlates With Renal Damage and Its Intercellular Communication Role in Systemic Lupus Erythematosus. Frontiers in Immunology, 12, 630112.

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