A30P 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. The base medium for this cell line is Advanced DMEM/F12 (Gibco;12634010). To make the complete growth medium

add the following components to the base medium: N2 supplement (ThermoFisher Scientific) to a final concentration of 1X

L-glutamine (G275) to a final concentration of 2 mM

and Recombinant Human FGF2 (Z101455) to a final concentration of 40 ng/ml. Cells may be grown in the presence of 1 μg/ml Tetracycline. Change media every 2-3 days. Do not let media colour change to orange-yellow. The cells will form round clumps instead of a monolayer when stressed. It is recommended to subculture when cells are grown to a cell density of 50-60%. Carbon dioxide (CO2): 5%

Temperature: 37.0°C. 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.