Multiskan rc microplate reader

Cell viability and proliferation were evaluated by MTT assay. Thousand cells in 100 μl of medium were seeded per well into 96-wells plate and then incubated for 1, 3, 5 and 7 days, and was replaced with fresh medium after every two days. After incubation, medium was discarded and supplied with 100 μl of MTT solution (Sigma, Saint Louis, MO, USA), then incubated at 37°C for 4 hours. After 4-hours incubation, MTT solution was removed and 50 μl of DMSO was added to dissolve the dark blue formazan crystal, and absorbance was determined using ELISA reader (Multiskan RC Microplate Reader, Thermo) at 595 nm.

Mice were sacrificed by cervical dislocation, the brains were rapidly removed, and the striatum and midbrain containing substantia nigra (SN) were dissected. Tissue samples were weighed and homogenized in 20 volumes of ice-cold homogenization buffer to wet tissue weight. After centrifugation, protein concentrations in the supernatants were determined using Protein Assay Dye Reagent Concentrate (Bio-Rad). Enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-1β (ab100705, Abcam), GFAP (NS830, Merck), BDNF (CYT306, Merck), and GDNF (e0043m, EIAab) concentrations in the brain regions of interest. All steps of quantification were performed according to the manufacturer's recommendations. Standards and samples were added to the microtiter plate in triplicate, the optical density of each well was determined at 450 nm (Thermo LabSystems, Multiskan RC Microplate Reader), and protein concentrations were calculated from the standard curve. Results were expressed in pg/ml for BDNF, GDNF, and Il-1β or in ng/ml for GFAP.

Cell viability was evaluated through MTT assay, in which 1 × 10³ cells/well of ihNPs were seeded into 96-well plate, then incubated in high-glucose medium (HGM), with or without PDB treatment (TGF-β1 = 0.1 or 2 ng/mL), for 1, 3, 5 and 7 days; the medium was replaced every two days. Thereafter, the medium was discarded and replaced with 100 μL of MTT solution (Sigma, Saint Louis, MO, USA), then incubated for 4 h at 37 °C. Next, MTT solution was replaced with DMSO to dissolve the dark blue formazan crystal, and the absorbance was measured at 595 nm using ELISA reader (Multiskan RC Microplate Reader, Thermo). For evaluating cell proliferation, 5 × 10⁵ cells/well of ihNPs were seeded into six-well plates, then incubated in control (DMEM/F12 + 10% FBS), glucose, or glucose + PDB medium according to their experimental groups. After 5 days of treatment, cell numbers of each group were determined through the automated cell counter Countess™ (Life Technologies, Carlsbad, USA).