Chemikine bdnf sandwich elisa kit

After lethal pentobarbital injection, rats were craneotomized and brain samples were rapidly excised. The hippocampus was dissected, weighed, and snap frozen in liquid nitrogen prior to storage at -70°C. After four weeks of freezing, tissue samples were homogenized in ice-cold homogenization buffer consisting of 100mM Tris/HCl (pH 7), 2% bovine serum albumin (BSA), 1M NaCl, 4mM EDTA, 2% Triton X-100, 0.1% NaN₃, and the following protease inhibitors: 5μg/mL aprotinin, 0.5μg/mL antipain, 157μg/mL benzamidine, 0.1μg/mL pepstatin A, and 17μg/mL phenylmethyl-sulphonyl fluoride. Homogenates were prepared in approximately 20 volumes of the homogenization buffer to tissue-wet weight and centrifuged at 14,000g for 30 minutes. The resulting supernatants were used for the BDNF assay. Samples were analyzed by triplicate, following the instructions of the ChemiKine^™ BDNF Sandwich ELISA Kit (Millipore, USA). Absorbance was measured in a microplate spectrophotometer at a wavelength of 450nm (MultiSkan, Thermo Scientific, Finland).

Both stimulated and unstimulated cells were cultured in T25 flask (Nunc). A final volume of 8 ml of conditioned media was collected. The cell numbers were calculated (≈8 × 10⁶ ± 2 × 10⁵ cells/group). The conditioned media were analysed by ELISA using the ChemiKine™ BDNF sandwich ELISA kit (Millipore) or NGF, GDNF, angiopoietin-1 and VEGF-A sandwich ELISA kits (RayBiotech Inc) according to the manufacturer’s protocol and as previously described^{7 (link)}. All samples were analysed in triplicate and the absorbance was measured at 450 nm on a SpectraMax190 microplate reader (Molecular Devices Inc). The quantity of factors (pg/ml) were calculated against standard curves produced using recombinant proteins provided in the kits and normalised to the final number of cells (1 ml conditioned media from 1 × 10⁶ cells). Intracellular production of one of the proteins, BDNF, was confirmed by immunostaining. In brief, after fixation and blocking with normal goat serum, rabbit anti-BDNF antibody (1:50, Santa Cruz Biotechnology) was applied to cell cultures for 2 h, followed by washing and application of secondary Alexa Fluor 568 conjugated goat anti-rabbit IgG (1:1000; Molecular Probes) for 1 h at room temperature in the dark. The slides were cover slipped with Prolong anti-fade mounting medium containing 4′-6-diamido-2-phenylindole (DAPI, Invitrogen).