Wild Type aSYN-IRES-GFP Stable LUHMES Cell Line

Grow the cells in culture vessel pre-coated with 50 μg/ml Poly-L-Ornithine (PLO) (TM062) and 1 μg/ml fibronectin (EMD Millipore; Cat. FC010) in H2O for at least 3 hours at 37°C. Do not grow these cells in culture vessels with surface areas equal to or less than 12.5 cm². These cells do not grow in 6-well

24-well

48-well

or 96-well plates. Advanced DMEM/F12 (Gibco;12634010) + 1X N2 supplement (ThermoFisher Scientific) + 2 mM L-glutamine + 40 ng/ml recombinant bFGF (Z101455) + 1% Penicillin/Streptomycin Solution (G255)

37.0°C

5% CO₂. Cells may be grown in the presence of 1 μg/ml Tetracycline. To subculture the cells

use TrypLE Express. After adding TrypLE Express

incubate the cells at 37.0°C incubator for 2-3 minutes and agitate the culture vessel until 90% of the cells have detached. Immediately neutralize the trypsin using Trypsin Neutralizing Solution (Lonza

Cat. CC-5002). Add Trypsin Neutralizing Solution the same volume of trypsin used. Centrifuge at 200x g for 2-3 minutes. Aspirate supernatant and resuspend cells in complete media. Plate cells into pre-warmed PLO-fibronectin-coated vessels at 37.0°C. Avoid subculturing if the cells appear stressed. Note: If leaving the cells over the weekend (or more than 2 days)

make sure to do a high split ratio (1:4 to 1:5). For information regarding cell differentiation

please refer to the Differentiation Protocol PDF under the Documents Tab.