Mouse TNF-alpha Antibody

Detection of TNF-alpha in Raw264 Mouse Cell Line by Flow Cytometry. Raw264 mouse macrophage cell line treated with 1 μg/mL LPS overnight was stained with Rat Anti-Mouse TNF-alpha Monoclonal Antibody (Catalog # MAB4101, filled histogram) or isotype control antibody (MAB005, open histogram) followed by Anti-Rat IgG CFS-conjugated Secondary Antibody (F0104). To facilitate intracellular staining, cells were fixed and permeabilized with FlowX FoxP3/Transcription Factor Fixation & Perm Kit (FC012). Staining was performed using our Staining Intracellular Molecules protocol.

Detection of TNF-alpha in Mouse Splenocytes by Flow Cytometry. Mouse splenocytes (A) treated with PMA (50 ng/mL), Ca²⁺ Ionomycin (200 ng/mL) and Brefeldin A (5 μg/mL) for 4 hours or (B) untreated, were stained with Rat Anti-Mouse TNF-alpha Monoclonal Antibody (Catalog # MAB4101) followed by Anti-Rat IgG PE-conjugated Secondary Antibody (F0105B) and Rat anti-Mouse CD3 APC-conjugated Monoclonal Antibody (FAB4841A). Quadrant markers were set based on isotype control antibody (MAB005). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (FC005). Staining was performed using our protocol for Staining Intracellular Molecules.

Cytotoxicity Induced by TNF‑ alpha and Neutralization by Mouse TNF‑ alpha Antibody. Recombinant Mouse TNF-a (Catalog # 410-MT) induces cytotoxicity in the the L-929 mouse fibroblast cell line in a dose-dependent manner (orange line). Cytotoxicity elicited by Recombinant Mouse TNF-a (0.25 ng/mL) is neutralized (green line) by increasing concentrations of Mouse TNF-a Monoclonal Antibody (Catalog # MAB4101). The ND₅₀ is typically 0.15-0.75 µg/mL in the presence of the metabolic inhibitor actinomycin D (1 µg/mL).

Detection of Mouse TNF-alpha by Western Blot Parameters of inflammation/nitrosative stress are unaltered in adipose tissue after chronic administration of compound 21. (A) and (B) Levels of TNF‐ alpha determined by WB and IHC. (C) and (D) levels of nitrotyrosine determined by WB and IHC. Quantification of specific bands was performed with Gel‐Pro Analyzer software. (C) For nitrotyrosine quantification, the intensity of the most predominant bands in the WB (of unknown identity), with MWs of 53 and 65 kDa, respectively, were quantified in each sample. Bar graphs are the means ± SE. (B) and (D) Positive TNF‐ alpha and nitrotyrosine staining as detected by immunohistochemistry was quantified using Image‐Pro Plus software. Data were analyzed by unpaired two‐tailed Student's t test. C21, n = 9 per group, control, n = 12 per group for TNF‐ alpha quantification. For nitrotyrosine quantification by Western blotting: C21, n = 6 per group; control, n = 6 per group and by immunohistochemistry: C21, n = 9 per group; control, n = 9 per group. WB, western blotting. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/30156060), licensed under a CC-BY license. Not internally tested by R&D Systems.