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Immortalized Human Dopaminergic Neuronal Precursor Cells (LUHMES)

CAT.NOUNITPRICE
T02841x106 cells / 1.0 ml
$250.00
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Specifications


Description

The Immortalized Human Dopaminergic Neuronal Precursor Cells, also known as the Lund Human Mesencephalic (LUHMES) cell line, is a subclone of the tetracycline-controlled, v-myc-overexpressing human mesencephalic-derived cell line MESC2.10. This cell line is unique in that it can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. LUHMES expresses functional dopamine transporter (DAT), vesicular monoamine transporter (VMAT-2), tyrosine hydroxylase (TH) and the neuronal form of β-III tubulin after differentiation. In addition to retaining dopaminergic neuronal-specific markers, LUHMES also exhibit electrophysiological properties, thus making this cell line a valuable neuronal model for neurodevelopmental studies, disease modelling and neuropharmacology.

SKUT0284
SpeciesHuman (H. sapiens)
Tissue/Organ/Organ SystemBrain
Donor Age8 weeks old
Growth PropertiesAdherent
Cell MorphologyFlattened|Dendritic processes
Seeding Density50,000 - 100,000 cells/cm2; cells will have viability of around 40-50%. Post thawing, it will need 4-5 days for cells to recover in culture; Recommended split ratio is 1:4 to 1:5
Population Doubling Time30 - 40 hours
Immortalization MethodConditional immortalization by tetracyclin-controlled transduction with retrovirus carrying v-myc genes
Applications

For Research Use Only

Unit quantity1x106 cells / 1.0 ml
Cell TypeImmortalized Cells
Expression Profile

DAT, VMAT-2, TH, α-SYN, β-III tubulin

Propagation Requirements

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.
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.

QC

mRNA and protein expression levels of various markers pre and post differentiation are verified by RT-PCR and western blotting.

Disclaimer

1. For for-profit organizations and corporations, please contact [email protected] for pricing of this item.

2. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at [email protected].

3. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final.

4. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping).

5. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information.

6. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.

7. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period."

DepositorUniversity of Konstanz
Documents


Supporting Protocol
MSDS

    QC

    Other
    FAQs


    I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?
    We do not carry out downstream characterization or gene expression profiling of our cell lines. To facilitate your preliminary experiments we can provide an RNA extraction (0.5ug total RNA) or cell lysate (100ug/100ul provided in 62.5mM Tris‐HCl, 2% SDS, 10% Glycerol, 50mM DTT, 0.01% w/v Bromophenol Blue) for any of our immortalized cell lines for a small fee. Please inquire directly for more information. The lead time will be around 2 weeks from the time of placing an order (if the item is in stock).
    How often do I need to change the media?
    The media should be changed every 2-3 days.
    Why do these cells need bio safety level II?
    In order to be more cautious, we follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site if required.
    Do you sell ECM coated T75 flasks?
    Yes we can provide a coating service. Please inquire with [email protected]
    Is it necessary that we have to use the recommended T25 ECM-coated flasks for growing the cells? Can we use normal ECM coated 60mm/90mm petri plates for growing the cells?
    We strongly recommend using G299 T25 flasks to ensure recovery of these cells before testing other plates.
    What can I coat a larger dish to subculture?
    We also offer applied extracellular matrix (collagen type I) in liquid form, for the coating of larger flasks and other required plasticware: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html
    How long can I store frozen vials for?
    Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen indefinitely without affecting their recovery.
    When are cells plated for live cell shipments?
    1 day prior to shipping
    Should the cap of the flask be changed before starting the cell culturing step?
    No, there is no need in sterile biosafety cabinets unless it has contacted any non-sterile condition (e.g. touching the contaminated tip, etc.).
    What is the recommended storage temperature?
    In general, if you received: Live cells: acclimatize for 3-4 hrs at at the recommended conditions stated for the cell line under the propagation section, and then change media afterwards. Frozen cells: Immediately place cells in liquid nitrogen; -180C.
    Differentiated cells are to be grown on PLL coated plates. What kDa range of these polymers do you suggest for coating?
    We recommend PLO coating (MW 30,000-70,000).
    How is cell density crucial for drug selection?
    If antibiotic selection is applicable to the target cells, we suggest getting rid of all the background cells so that the cell density is kept lower (even 20-30%). However, once the clones are selected by clonal dilution, we don't need the drug to still be present. If needed, the cell density should be towards the higher end since cells are already selected. Any primary cells still present will be depleted as a result of senescence and the cell population that remains will be resistant to the specific antibiotic.
    My cells are not detaching, what method do you recommend to trypsinize the cells?
    1. Incubate the coated plate containing trypsin solution at recommended temperature indicated in the propagation section for 3-5 min till the cells round up, monitoring from time to time under microscope. 2. Diluting G422 (1:1) with PBS and coating for lesser time. Sometimes the collagen content in G422 is higher and thus make stronger bonding with cells. 3. You can try reducing the incubation time as well for coating the plate to make a thinner layer.
    Why is it important to determine the optimal seeding density?
    The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate. If you seed too little, cells may not attach well to the surface (for adherent cells). Seeding density is important as many cells (adherent or suspension cells) need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed. If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating.
    References


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