Through lentiviral invfection, the A53T aSYN-IRES-GFP Stable LUHMES Cell Line was generated. The cells stably express A53T aSyn. This cell line can be used to study neurotoxicity and to identify targets for treatment/prevention of neurodegenerative disorders.abm also offers A30P aSYN-IRES-GFP Stable LUHMES Cell Line (T6454), Wild Type aSYN-IRES-GFP Stable LUHMES Cell Line (T6456), and IRES-GFP Stable LUHMES Cell Line (T6457).
Quantity: 1 million cells/1.0 ml
Organism: Human (H. sapiens)
Source Organ: Brain
BioSafety Level: II
Growth Properties: Adherent
Morphology: Epithelial
Markers: A53T aSyn, GFP, Tetracycline resistance
Applications: For Research Use Only
Propagation: 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 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 0.25% Trypsin-EDTA (TM051). After adding trypsin, incubate the cells at room temperature 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 twice the 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). To differentiate the cells into neurons, change the medium to differentiation medium after the cells have grown to a density of 40-50%.To make complete differentiation medium, add the following components to Advanced DMEM/F12 (Gibco;12634010): N2 supplement to a final concentration of 1X, L-glutamine (G275) to a final concentration of 2 mM, dbcAMP to a final concentration of 1 mM, tetracycline to a final concentration of 1 μg/ml, and Recombinant Human GDNF (Z101055) to a final concentration of 2 ng/ml. Allow the cells to grow in differentiation medium for 4-6 days before testing for neuron specific markers.
Quality Control: 1) Western blot; 2) Immunocytochemistry
Shipping Conditions: Dry Ice
Storage Conditions: -180°C