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FluoroSpot MAX™ Deluxe Set Human TNF-α/IFN-γ

Pricing & Availability
Regulatory Status
RUO
Other Names
Tumor Necrosis Factor α, (TNF α), Cachectin, Cytotoxic Factor (CF), Hemorrhagic Factor
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Product Citations
publications
a. FluoSpot_Human-TNF-g-IFN-y_1_073025
Human PBMC (5,000 cells / well) were incubated for 22.5 hours in the presence of 100 ng/mL LPS. The yellow spots represent IFN-γ (PE) secreted cells and red spot represent TNF-α (APC) secreted cells. The number of cells secreting Human TNF-α/IFN-γ was analyzed by FluoroSpot and quantified using an image cytometer equipped with the required fluorescence filters.
  • a. FluoSpot_Human-TNF-g-IFN-y_1_073025
    Human PBMC (5,000 cells / well) were incubated for 22.5 hours in the presence of 100 ng/mL LPS. The yellow spots represent IFN-γ (PE) secreted cells and red spot represent TNF-α (APC) secreted cells. The number of cells secreting Human TNF-α/IFN-γ was analyzed by FluoroSpot and quantified using an image cytometer equipped with the required fluorescence filters.
  • b. FluoSpot_Human-TNF-g-IFN-y_2_073025
    Human PBMC (5,000 cells/well) were incubated for 5 hours in the absence or presence of either 1X Cell Activation Cocktail without Brefeldin A (BioLegend, Catalog No. 423301) or 1X Cell Activation Cocktail with Brefeldin A (Cat. No. 423301) or 1X Cell Activation Cocktail with Brefeldin A (Cat. No. 423303). All figures showed the merged figure of red and Far-red. Spot numbers were counted for double-positive spots. BioLegend’s Cell Activation cocktail contains PMA and ionomycin at the optimized concentration. Brefeldin A functions to inhibit intracellular protein transport, as a result, it inhibits secretion of cytokines such as TNF-α and IFN-γ. The number of cells secreting Human TNF-α/IFN-γ was analyzed by FluoroSpot and quantified using an image cytometer equipped with the required fluorescence filters.
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Description

Human TNF-α (Tumor Necrosis Factor α), also known as Cachectin, Cytotoxic Factor (CF), is a cytokine which has important roles in regulating a wide range of biological functions from immune activation, cellular differentiation, proliferation, apoptosis, and energy metabolism. TNF-α is expressed and secreted by macrophages, neutrophils, T-cells, NK cells, and various other cell types. TNF-α can bind to two receptors: TNFR1 and TNFR2. Signaling through TNFR1 is mostly pro-inflammatory, while TNFR2 signaling is mainly anti-inflammatory and proliferative. Because of its diverse functions, dysregulation or TNF-α expression and activity has been implicated in many diseases from autoimmune, cancer, to neurodegeneration.

Human IFN-γ (Interferon-gamma) is a multifunctional cytokine which is the sole member of the class II Interferon. Although a variety of immune cells can express IFN-γ, most of IFN-γ is produced by T cells and NK cells. It has critical roles in many processes including, but not limited to, anti-viral response, regulating Th1/Th2 balance, enhancing antigen presentation, and controlling cellular proliferation and apoptosis. IFN-γ can bind to 2 receptors: IFNGR1 and IFNGR2, which transduce the signal downstream through the JAK-STAT pathway. Because of its multifunctional nature, dysregulation of IFN-γ expression and activity has been implicated in many diseases such as autoimmune and cancer.

BioLegend’s FluoroSpot MAX™ Deluxe Set contains pre-optimized essential reagents and buffers for the simultaneous detection of human TNF-α and IFN-γ secreting cells. In this assay, anti-human TNF-α and IFN-γ capture antibodies are coated on a clear-bottom microplate. Cells (stimulated or unstimulated) are added to the wells and incubated, allowing secreted TNF-α and/or IFN-γ to bind to nearby capture antibodies. After incubation, cells are removed, and fluorescent conjugated detection antibodies are added. The resulting spots represent the "footprints" of individual TNF-α and IFN-γ-secreting cells, enabling precise quantification of both single cytokine-producing cells and dual cytokine-producing cells within the sample.

Product Details
Technical Data Sheet (pdf)

Kit Details

Kit Contents
  • Human TNF-α (2-plex) Capture Antibody (200X) 
  • Human IFN-γ (2-plex) Capture Antibody (200X) 
  • APC Human TNF-α Detection Antibody (200X) 
  • PE Human IFN-γ Detection Antibody (200X) 
  • Human TNF-α Standard (used as a positive control) 
  • Human IFN-γ Standard (used as a positive control) 
  • Coating Buffer A (5X) 
  • Assay Diluent B (5X)
Materials Not Included
  • Flat Bottom High Bind Microplate: Corning® Clear Bottom Black Polystyrene Microplates, 96 Well (Catalog No. 3601) is recommended, or equivalent.
  • Black Microplate Lid: Corning® Universal Black Microplate Lid with Corner Notch (Catalog No. 3935) is recommended, or equivalent
  • Wash Buffer: BioLegend Cat. No. 421601 is recommended, or PBS + 0.05% Tween-20
  • Plate Sealers: BioLegend Cat. No. 423601 is recommended
  • 0.22 μm filters
  • Sterile PBS (Phosphate-Buffered Saline): 8.0 g NaCl, 1.16 g Na2HPO4, 0.2 g KH2PO4, 0.2 g KCl, add deionized water to 1 L; pH to 7.4, 0.22 μm filtered
  • Sterile cell culture media
  • 37°C CO2 incubator
  • Deionized (DI) water
  • Adjustable pipettes to measure volumes ranging from 2 μL to 1 mL 
  • Wash bottle, manifold dispenser, or automated microplate washer 
  • Tubes for dilution
  • Timer
  • Alcohol Wipes 
  • Image cytometer or other imaging platform capable of whole-well imaging. The device should be equipped with the appropriate filters for the fluorophores utilized in each assay.
  • Optional: Carboxyl Beads B4 (Cat. No. 740175 or equivalent)

Product Details

Verified Reactivity
Human
Application

FluoroSpot

Antigen Details

Cell Type
B cells, Eosinophils, Macrophages, Mast cells, Neutrophils, NK cells, NKT cells, T cells, Th1
Biology Area
Cancer Biomarkers, Cell Biology, Immuno-Oncology, Immunology, Innate Immunity, Neurodegeneration
Molecular Family
Cytokines/Chemokines, Innate Immune Signaling
Gene ID
7124 View all products for this Gene ID 3458 View all products for this Gene ID
UniProt
View information about TNF-alpha, IFN-gamma on UniProt.org

Related FAQs

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Go To Top Version: 1    Revision Date: 07/17/2025

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

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