Rat tnf α

In vitro procedures for the human astrocytoma cell line and primary rat astrocytes were similar except for the dosage of TNF-α (2000 IU/mL of human TNF-α produced at the Dept. of Biomedical Research of UGent) and 10 ng/mL rat TNF-α (Sigma-Aldrich, St. Louis, MO, USA), clenbuterol (Sigma-Aldrich) was administered at 10 μM for human and rat astrocytes. After 2 h of starvation on DMEM/1% FCS/Pen-Strep, cells were exposed to the different stimuli for 3 h: vehicle, clenbuterol, TNF-α, and TNF-α + clenbuterol. Cells were washed with ice-cold PBS and resuspended in 350 μL Qiagen RNeasy lysis buffer (RLT + β-ME) before homogenization with the Qiashredder. Ethanol (350 μL) was added and lysates were stored at -80°C until further processing.

BR, the bovine serum albumin (BSA), rat TNF-α, LPS from Escherichia coli 0114:B4, human insulin solution, Williams’ E Medium, Collagen type I from rat tail tendon, 2,6-di-tert-butyl-4-methylphenol (BHT), Thiazolyl Blue Tetrazolium Bromide (MTT), RNAlater, and tetrabutyl-ammonium hydroxide (TBA, 40 % in water) were purchased from Sigma-Aldrich (St. Louis, MO, USA); the chloroform (HPLC grade), methanol (HPLC grade), n-hexane, ethyl acetate, and acetonitrile were purchased from Merck (Darmstadt, Germany); and 4×-Laemmli sample buffer was from Bio-Rad (Hercules, CA, USA).
As described earlier, the BR was purified before use [40 (link)]. For the experiments, BR (2.8 mg) was dissolved in 2 mL of 0.1 M NaOH and immediately mixed with 1 mL of 0.1 M phosphoric acid. The mixture was diluted with a BSA solution (660 µM BSA in 25 mM phosphate buffer, pH: 7.7) to reach a final concentration of 480 µM BR in a phosphate buffer and then serially diluted with a BSA solution to yield solutions with final BR concentrations within the range of 10–40 µM.