FluoroSpot Path: Human immunotherapy potency (3‑color)

FSP-013609-TD-2

Publications: 0

Documents

Bulk or custom format

Paves your way to functional analysis of a specific research question.

Components

Plate	Pre-coated FluoroSpot plate (mAbs 1-D1K, MT34B6, and MT25C5)
Detection mAbs	Anti-IFN-γ mAb (7-B6-1), BAM
	Anti-Granzyme B (MT28), biotin
	Anti-TNF-α mAb (MT20D9), WASP
Fluorophore conjugates	Anti-BAM mAb, 490
	SA-550
	Anti-WASP mAb, 640
Buffer/Solution	FluoroSpot enhancer
Positive control	Anti-CD3 mAb (CD3-2)
Co-stimulus	Anti-CD28 mAb (CD28-A)

In stock

Delivery 4-9 business days

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Complementary products

1 / 2

PepPool: CEF (CD8), human

Product specifications

Intended use

This FluoroSpot Path kit is intended for the enumeration of cells secreting human IFN-γ, granzyme B, and TNF-α using the FluoroSpot assay. An orthogonal method for potency assays and MOA evaluation for cell therapy products. For research use only. Not for use in diagnostic procedures.

Serum/Plasma samples

Recommendation

Product details

Product	FluoroSpot Path: Human immunotherapy potency (3-color)
Application	FluoroSpot
Analyte	IFN-γ, Granzyme B, TNF-α
Reactivity	Human
Specificity	This kit is based on matched pairs of mAbs specific for human IFN-γ, granzyme B, and TNF-α.

Shipping and Storage

Shipping	Shipped at ambient temperature.
Storage	Store antibodies, fluorophore conjugates, and FluoroSpot enhancer at 4-8 °C upon receipt. Plates may be kept at room temperature.
Shelf life	At least 12 months from date of receipt.

Performance

PBMC (250,000 cells/well) were incubated for 42 hours in the absence or presence of a mixture of peptide pools: CEFTA peptide pool for human CD4 T cells (product code 3617-1), CEF extended peptide pool for human CD8 T cells (product code 3618-1), and CMV peptide pool for human CD4 and CD8 T cells (product code 3619-1) and anti-CD28 antibody. The numbers of cells secreting IFN-γ, Granzyme B and TNF-α was analyzed by FluoroSpot. Single cytokine secreting cells as well as double- and triple-secreting cells were determined. Spot center was used to identify spots from double- and triple-secreting cells, visualized in an image overlay of LED490, FITC (IFN-γ) and LED550, Cy3 (Granzyme B) and LED640, Cy5 (TNF-α). Spot analysis was made with Mabtech IRIS.

Documents

Datasheet/Protocol SDS

Tutorials

Tutorial, Published September 7, 2023

Step-by-step guide to FluoroSpot

An in-depth guide to the FluoroSpot protocol, including best practices and tricks we've learned over the years.

Tutorial, Published November 24, 2022

Cell stimulation in ELISpot and FluoroSpot

In this feature, we’ll walk you through the optimal use of different polyclonal and antigen-specific stimuli.

Tutorial, Published September 2, 2022

Isolation, freezing, and thawing of PBMCs

This is a protocol on how to best isolate, freeze, and thaw PBMCs.

Publications (0)

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Analyte information

IFN-γ

Analyte description	Interferon-γ (IFN-γ) is the only type II interferon. This proinflammatory cytokine is secreted by activated T cells and NK cells. It activates macrophages and endothelial cells and regulates immune responses by affecting APCs, T cells, and B cells. Production of IFN-γ by helper T cells and cytotoxic T cells is a hallmark of the Th1-type phenotype. Thus, high-level production of IFN-γ is typically associated with effective host defense against intracellular pathogens.
Alternative names	Interferon-γ, Interferon-gamma, IFN-γ, IFN-gamma, IFN-g, IFNg, IFG, IFI, IMD69
Cell type	T cell, Tc, Th1, NK cell
Gene ID	3458

Granzyme B

Analyte description	Granzyme B is a serine protease released by the cytoplasmic granules within cytotoxic T cells and NK cells. Granzyme B will act together with perforin and other cytotoxic mediators to induce target cell death by apoptosis.
Alternative names	Granzyme B, GzmB, GrB, GrzB, C11, CCPI, CGL-1, CGL1, CSP-B, CSPB, CTLA1, CTSGL1, HLP, SECT
Cell type	T cell, Tc, Th1, NK cell
Gene ID	3002

TNF-α

Analyte description	Tumor necrosis factor (TNF), also known as TNF-α, is produced by many different cell types, e.g., monocytes, macrophages, T cells, and B cells. Among the many effects of TNF-α are protection against bacterial infection, cell growth modulation, immune system regulation, and involvement in septic shock.
Alternative names	Tumor necrosis factor-α, TNF-α, TNF-alpha, TNF-a, TNFa, Tumor necrosis factor-alpha, TNF, DIF-alpha, TNFA, TNFSF2, TNLG1F
Cell type	T cell, Tc, Th1, Th2, Th17, Tfh, Monocyte/MΦ
Gene ID	7124