Anti mouse tnf α

Cell culture supernatants collected at 48h post infection were aliquoted into 200 μL volumes and frozen at -80°C. An ELISA test was conducted for IFN-γ, IL-6 and TNF-α according to the manufacturer’s instructions (R&D Biosystems). Briefly, wells of Nunc Maxisorp 96-well plates were coated with anti-mouse IL-6 (2 μg/ml), anti-mouse IFN-γ (4 μg/ml) or anti-mouse TNF-α (0.8 μg/ml) (R&D BioSystems) overnight at room temperature. Before use, the plates were blocked with PBS containing 1% bovine serum albumin fraction V (BSA, Sigma) for 2 h at room temperature. Cell culture supernatant samples were added to the wells in a volume of 100 μl and the control samples were diluted in PBS 1% BSA. After the 2 h incubation at room temperature, wells were washed 4 times with PBS containing 0.05% Tween 20. The addition of the biotinylated monoclonal antibodies for each of the cytokines (IL-6 150ng/ml; IFN-γ 300ng/ml; TNF-α 50ng/ml) were added and incubated for 2 h at room temperature. Horseradish peroxidase-conjugated streptavidin (R&D Biosystems) was added according to the manufacturer’s recommendations and incubated for 30 min at room temperature. Standard curves were generated using purified recombinant IL-6, IFN–γ and TNF-α according to the manufacturer’s recommendations (R&D BioSystems) using MaxPro generated 4-parameter curve-fit for each cytokine.

The anti-Lyn, anti-Src, anti-α-tubulin antibodies and horseradish peroxidase conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-mouse TNF-α, IL-6, and IL-1β ELISA kits were obtained from R&D Systems (Minneapolis, MN). Anti-phospho-tyrosine (4G10) antibody was from Upstate (Temecula, CA), and anti-pLyn (Tyr 396) antibody was purchased from Abcam (Cambridge, MA). Anti-pSrc, Src, pERK, pJNK, p-p38, JNK, p38 and ERK2 antibodies were purchased from Cell Signaling Technology (Danvers, MA). LPS (Lipopolysaccharides from E.Coli 026:B6) was obtained from Sigma-Aldrich Corp. (St. Louis, MO). The LDH cytotoxicity assay kit was from Promega Corporation (Madison, WI). Human Aβ1–42 was purchased from rPeptide (Bogart, GA).