Anti tnf α pe cy7

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The Anti-TNF-α-PE-Cy7 is a fluorochrome-conjugated antibody that binds to the tumor necrosis factor alpha (TNF-α) protein. It is designed for use in flow cytometry applications to detect and quantify TNF-α-expressing cells.

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TNF-α (2204 citations) Anti-TNF-α (42 citations) TNF-α-PE (12 citations) TNF-α-PE-Cy7 (9 citations) TNF-PE-Cy7 (2 citations)

7 protocols using anti tnf α pe cy7

Multicolor Flow Cytometry Immunophenotyping

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anti-CD14-BV570 (301831, Biolegend), anti-CD11c-PerCP-Cy5.5 (337210), anti-HLA-DR-AlexaFlour700 (307626) all from Biolegend, anti-CD66a/c/e-QDot605 (342302, Biolegend, with in-house conjugation to QDot605, Q-22001), anti-CD16-PE-Cy5 (555408, from BD Biosciences), anti-IL-12-PE (554575), anti-TNF-α-PE-Cy7 (25-7349-82) both from eBioscience, anti-IL-10-PE (506804), anti-IL-6-APC (501112), anti-CD3-BV421 (300434), anti-CD19-BV421 (302233) and anti-CD335-BV421 (331913) all from Biolegend.
Cryopreserved, fixed white cells were washed in PBS and stained for 1 hour at 4°C with surface marker antibodies. For intracellular staining, cells were permeabilized with Perm/Wash buffer (BD Biosciences) and incubated for 1 hour at 4°C with antibodies for intracellular markers. Fixed cells were then acquired on a LSR II flow cytometer (BD Biosciences).

Baguma R., Penn-Nicholson A., Smit E., Erasmus M., Day J., Makhethe L., de Kock M., Hughes E.J., van Rooyen M., Pienaar B., Stone L., Hanekom W., Brennan M.J., Wallis R.S., Hatherill M, & Scriba T.J. (2017). Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population. PLoS ONE, 12(9), e0184563.

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Neoantigen-Specific T-cell Response Quantification

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The neoantigen-specific T-cell response was determined using intracellular cytokine staining (ICS) performed by FC detection and IFNγ ELIspot as previously described^{19 (link)} For the FC analysis, the following antibodies were utilized according to different panels: anti-CD3-Alexafluor488 (cat. 53–0031-82, eBioscience), anti-CD3-PEefluor610 (cat. 61–0031-82, eBioscience), anti-CD4-PerCP-Cy5.5 (cat. 45–0042-82, eBioscience), anti-CD8-APCeFluor780 (cat 47–0081-82, eBioscience), anti-CD45-efluor450 (cat. 48–0451-82, eBioscience), anti-CD45-APC (cat. 559,864, BD), NK1.1-BV786 (cat. 740,853, Bio Legend), anti-CD11b-FITC (cat. 53,310, BD), anti-CD11c-SB645 (cat. 64–0114-82, Ebioscience), anti-LY6C-PE-Cy7 (cat. 560,593, BD), anti-LY6G-Alexafluor700 (cat. 561,236, BD), anti-IFN-γ-PE (cat. 12–7311-82, eBioscience), anti-TNF-α-PE-Cy7 (cat. 25–7321-82, eBioscience), anti-TNF-α-eFluor450 (cat. 48–7321-82, eBioscience FC was carried out with a Citoflex flow cytometer (Beckman Coulter) and data analyzed with Cytexpert software (Beckman Coulter). For the IFNγ ELIspot cells were plated at 4 × 10⁵ and 2 × 10⁵ cells/well in duplicate and spots were counted using an automated ELISPOT reader (Aelvis ELIspot reader, A.EL.VIS Gmbh, Germany).

Lione L., Salvatori E., Petrazzuolo A., Massacci A., Maggio R., Confroti A., Compagnone M., Aurisicchio L., Ciliberto G, & Palombo F. (2021). Antitumor efficacy of a neoantigen cancer vaccine delivered by electroporation is influenced by microbiota composition. Oncoimmunology, 10(1), 1898832.

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Fluorochrome-Conjugated Antibodies for Immunostaining

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Fluorochrome-conjugated antibodies used: anti-CD11c-APC, anti-IFNγ-APC, anti-TNFα-PE-cy7, anti-IL-4-PE, anti-IL17-PE, anti-Foxp3-PE (e-Bioscience, Vienna, Austria), and anti-LAMP-1-v450 (BD-Pharmingen). Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le^X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova). Secondary antibodies used: peroxidase-labeled F(ab’)₂ fragment goat anti-human IgG, F(ab’)₂ fragment goat anti-mouse IgG, (Jackson), peroxidase-labeled goat anti-mouse IgM (Nordic Immunology), goat anti-rat Alexa 448, goat anti-rat Alexa 647, donkey anti-goat Alexa 488, donkey anti-goat Alexa 647, donkey anti-rabbit Alexa 555 and donkey anti-rabbit Alexa 488 (Molecular Probes). MGL-1-Fc was generated as described earlier (Singh et al., 2009b (link)). MR-Fc was kindly provided by L. Martinez-Pomares (University of Nottingham, Nottingham, UK).

Streng-Ouwehand I., Ho N.I., Litjens M., Kalay H., Boks M.A., Cornelissen L.A., Kaur Singh S., Saeland E., Garcia-Vallejo J.J., Ossendorp F.A., Unger W.W, & van Kooyk Y. (2016). Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells. eLife, 5, e11765.

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Multi-parameter Immunophenotyping of T Cells

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The following human monoclonal fluorescently-conjugated antibodies were used in this study: anti-CD3 Pacific Blue (clone UCHT1), anti-CD3 APC-H7 (clone SK7), anti-CD4 APC (clone RPA-T4), anti-CD8 PerCP-Cy5.5 (clone SK-1), anti-IFN-γ Alexa Fluor 700 (clone B27), anti-IL-2 FITC (clone 5344.111), anti-IL-2 APC (clone MQ1-17H12), anti-CCR7 FITC (clone 150503), anti-CD45RA PE-Cy7 (clone HI100), all from BD Biosciences, and anti-CD4 QDot605 (clone S3.5; Life Technologies), anti-TNF-α PE-Cy7 (clone Mab11; eBiosciences), and anti-PD-1 PE (clone EH12.2H7; BioLegend). For PD-1/PD-L1 blockade experiments, LEAF™ purified anti-human PD-L1 Ab (clone 29E.2A3) and LEAF™ purified IgG2b isotype-matched control Ab (clone MPC-11) were obtained from BioLegend.

Day C.L., Abrahams D.A., Bunjun R., Stone L., de Kock M., Walzl G., Wilkinson R.J., Burgers W.A, & Hanekom W.A. (2018). PD-1 Expression on Mycobacterium tuberculosis-Specific CD4 T Cells Is Associated With Bacterial Load in Human Tuberculosis. Frontiers in Immunology, 9, 1995.

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Multiparameter Flow Cytometry of CD8+ T Cell Subsets

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To analyze the frequency of CD8+ T cell subsets, the harvested cells were washed once with PBS and evaluated by flow cytometry. In brief, the 10⁶ cells/100ul staining buffer (PBS + 2%FCS) were first stained with Amcyan-conjugated anti-human CD8 monoclonal antibodies (BD Biosciences, US) for 30 minutes in the dark at 4°c. After surface staining, the cells were fixed and permeabilized using LEUCOPERM^™ (BIO-RAD, BUF09B, US) according to the manufacturer’s instructions. The cells were stained with anti-IFN-γ-FITC, anti-TNF-α-PE-Cy^™7, anti-IL-10-eFluor^®450, anti-IL-17-PE, anti-IL-21-eFluor^®660, anti-IL-22-PerCP-eFluor^®710 (eBioscience, US) and anti- IL-4-APC-Cy^™7 (BioLegend, US).

Salehi Z., Doosti R., Beheshti M., Janzamin E., Sahraian M.A, & Izad M. (2016). Differential Frequency of CD8+ T Cell Subsets in Multiple Sclerosis Patients with Various Clinical Patterns. PLoS ONE, 11(7), e0159565.

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Intracellular Cytokine and CD107a Analysis of NK Cells

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To measure the expression levels of intracellular cytokines and CD107a by NK cells, NK cells were co-cultured with tumor targets at an E:T ratio of 1:1 for 4 h in the presence of anti-CD107a-allophycocyanin (APC) (BD Biosciences), monensin (GolgiStop; BD Biosciences, Eugene, OR, USA), and brefeldin A (GolgiPlug; BD Biosciences). After 4 h, cells were washed with fluorescence-activated cell sorter (FACS) flow buffer (BD Biosciences) and stained with anti-CD3-FITC (BD Biosciences), anti-CD56-APC-eFluor®780 (BD Biosciences), and 7-aminoactinomycin (BD Biosciences). Subsequently, the samples were permeabilized by BD CytoFix/CytoPerm^TM (BD Biosciences) and stained with anti-IFN-γ-PE (BD Biosciences) or anti-TNF-α-PE-Cy7 (eBioscience). Stained cells were acquired on an LSR Fortessa and data was analyzed using FlowJo software (TreeStar Inc.).

Oh E., Min B., Li Y., Lian C., Hong J., Park G.M., Yang B., Cho S.Y., Hwang Y.K, & Yun C.O. (2019). Cryopreserved Human Natural Killer Cells Exhibit Potent Antitumor Efficacy against Orthotopic Pancreatic Cancer through Efficient Tumor-Homing and Cytolytic Ability (Running Title: Cryopreserved NK Cells Exhibit Antitumor Effect). Cancers, 11(7), 966.

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Quantifying T Cell Cytokine Production

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After isolation of PBMC and 5 h of in vitro activation with 50 ng/mL phorbol myristate acetate (PMA; Sigma Aldrich, Zwijndrecht, the Netherlands) and 1 μg/mL ionomycin (Sigma Aldrich) in the presence of 1.25 μg/mL monensin (BD Biosciences, Breda, the Netherlands), cells were stained to identify CD8 + T cells producing the pro-inflammatory cytokines TNFα and IFNγ. Hereto, they were stained for surface markers with anti-CD3-Horizon V450 and anti-CD8-APC-H7 (both BD Biosciences), and after fixation and permeabilisation (cytofix/cytoperm, BD Biosciences) the cells were stained intracellularly using anti-TNFα-PECy7 (eBioscience, Vienna, Austria) and anti-IFNγ-FITC (BD Biosciences). Cells were measured by flow cytometry (FACS Canto II flow cytometer, BD Biosciences) and analyzed with FACS Diva software (version 8.01, BD Biosciences).

Rolf L., Muris A.H., Bol Y., Damoiseaux J., Smolders J, & Hupperts R. (2017). Vitamin D₃ supplementation in multiple sclerosis: Symptoms and biomarkers of depression. Journal of the neurological sciences, 378.

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