P8340 1ml

The caudal quarter of the flash frozen brain tissue was homogenized in radioimmunoprecipitation assay buffer (1% Triton-X, 0.1% SDS, 1X PBS) and 1% phosphatase inhibitor cocktail (P5726-1ML, Sigma-Aldrich) and 1% protease inhibitor cocktail (P8340-1ML, Sigma-Aldrich). As previously described, the samples were sonicated on ice in 3s bursts until homogenized and then were centrifuged at 4°C [65 (link)]. The protein concentration of each sample was measured using Direct Detect Infrared Spectrometer. Each sample was stored at -80°C until the LUMINEX assay was performed. Cytokines and chemokines were assessed using the MILLIPLEX-MAP-Mouse-Cytokiine/Chemokine-Magnetic-Bead-Panel (MCYTOMAG-70K, EMD Millipore). This multiplex panel allows the detection of 25 different cytokines/chemokines and includes individual quality controls for each cytokine/chemokine. The samples were plated as duplicates and the plate was analyzed using a LUMINEX MAGPIX xPONENT 1.2 System which uses Milliplex Analyst software and Luminex technology to detect individual cytokine/chemokine quantities.

Cells were collected and washed three times with PBS. To obtain cell lysates, the cells were suspended in 1 ml cold whole cell lysis buffer (50 mM Tris–HCl, pH 7.5, 0.5 M NaCl, 1% NP-40, 0.1% SDS), supplemented with protease inhibitors (P8340-1 ML, Sigma Aldrich) and phosphatase inhibitors (P044-1 ML, Sigma Aldrich). The cytosolic protein fraction was isolated by centrifugation at 13,000g for 30 min at 4 °C to remove cellular debris. Protein concentration was determined using Bradford assay (Bio-Rad). An aliquot of 500 μg proteins from each sample was mixed with 4-fold volume cold acetone to precipitate proteins. All samples were centrifuged at 13,000g for 5 min. Precipitated proteins were collected and dried in SpeedVac concentrator. The pellet was resuspended in 500 μl of sodium phosphate buffer (pH 8.0), reduced in 10 mM DTT (1 h at 60 °C), alkylated with 30 mM iodoacetamide (20 min, room temperature in the dark), and digested with 10 μg trypsin (Promega) at 37 °C on orbital shaker overnight.

Homogenates from hippocampal tissue were prepared by lysis in immunoprecipitated buffer (RIPA - Thermo Scientific, 89901) with protease inhibitor cocktail (P8340-1ML, Sigma-Aldrich). Lysates were sonicated for 10 s, placed on ice for 20 min, and centrifuged 15 min at 14,000 rpm at 4 °C. Lysate supernatants were saved for protein analyses. Pro-MMP9 protein concentration in hippocampal lysates was measured by ELISA, performed according to the manufacturer’s protocol (Mouse Pro-MMP9, R&D systems, catalogue number MMP900B).