|Cytotoxicity Induced by TNF‑ alpha and Neutralization by Equine TNF‑ alpha Antibody. Recombinant Equine TNF‑ alpha (Catalog # 1814‑ET) induces cytotoxicity in the the L‑929 mouse fibroblast cell line in a dose‑dependent manner (orange line), as measured by Resazurin (Catalog # AR002). Cytotoxicity elicited by Recombinant Equine TNF‑ alpha (1 ng/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Equine TNF‑ alpha Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1814). The ND50 is typically 0.01-0.03 µg/mL in the presence of the metabolic inhibitor actinomycin D (1 µg/mL).|
|TNF‑ alpha in Equine PBMCs. TNF‑ alpha was detected in immersion fixed equine peripheral blood mononuclear cells (PBMCs) using Goat Anti-Equine TNF‑ alpha Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1814) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.|
Tumor necrosis factor alpha (TNF-alpha ) also known as cachectin and TNFSF2, is the prototypic ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, apoptosis, and immune system development. TNF-alpha is produced by a wide variety of immune and epithelial cell types (1, 2). Equine TNF-alpha consisits of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 178 aa extracellular domain (ECD) (3). Within the ECD, equine TNF-alpha shares 69%‑88% aa sequence identity with bovine, canine, cotton rat, feline, human, mouse, porcine, rat, and rhesus TNF-alpha. The 26 kDa type 2 transmembrane protein is assembled intracellularly to form a noncovalently linked homotrimer (4). Ligation of this complex induces reverse signaling that promotes lymphocyte costimulation but diminishes monocyte responsiveness (5). Cleavage of membrane bound TNF-alpha by TACE/ADAM17 releases a 55 kDa soluble trimeric form of TNF-alpha (6, 7). TNF-alpha trimers bind the ubiquitous TNF RI and the hematopoietic cell-restricted TNF RII, both of which are also expressed as homotrimers (1, 8). TNF-alpha regulates lymphoid tissue development through control of apoptosis (2). It also promotes inflammatory responses by inducing the activation of vascular endothelial cells and macrophages (2). TNF-alpha is a key cytokine in the development of several inflammatory disorders (9). It contributes to the development of type 2 diabetes through its effects on insulin resistance and fatty acid metabolism (10, 11).