Immortalized Human Dopaminergic Neuronal Precursor Cells (LUHMES)

Cat. No.

T0284

Unit

1x10⁶ cells / 1.0 ml

Price

$440.00

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Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing institution.

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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 licensing@abmgood.com.

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. May take 1.4 to 5 days for cells to recover in culture.
Cat. No.	T0284
Name	Immortalized Human Dopaminergic Neuronal Precursor Cells (LUHMES)
Unit	1x10⁶ cells / 1.0 ml
Price	$440.00
Category	Immortalized Cell Lines
Cell Type	Immortalized Cells
Organism	Human (H. sapiens)
Tissue	Brain
Donor History	8-week-old fetal human ventral mesencephalon
Cell Morphology	Flattened\|Dendritic processes
Growth Properties	Adherent, flat dendritic processes
Seeding Density (cells/cm²)	50,000 - 100,000
Split Ratio	1:4 to 1:5
Population Doubling Time	30 - 40
Immortalization Method	Conditional immortalization by tetracyclin-controlled transduction with retrovirus carrying v-myc genes
Expression Profile	DAT, VMAT-2, TH, α-SYN, β-III tubulin
Expression	DAT, VMAT-2, TH, α-SYN, β-III tubulin
Propagation Method	Grow cells in culture vessels 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 cells in culture vessels with surface areas equal to or less than 12.5 cm²; 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 (G275) + 40 ng/ml rh FGF2 (Z101455) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 5% CO₂. Do not let media colour change to orange-yellow. For information regarding cell differentiation, please refer to the Differentiation Protocol PDF under the Documents Tab.
Growth Conditions	Grow cells in culture vessels 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 cells in culture vessels with surface areas equal to or less than 12.5 cm²; 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 (G275) + 40 ng/ml rh FGF2 (Z101455) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 5% CO₂. Do not let media colour change to orange-yellow. For information regarding cell differentiation, please refer to the Differentiation Protocol PDF under the Documents Tab.
Subculture Protocol	Volumes given below are for a T75 flask; proportionally increase or decrease the volume as required per culture vessel size. Subculture cells once the culture vessel is 80% confluent. 1. Aspirate the culture media, and add 2-3ml of pre-warmed 0.25% Trypsin-EDTA to the culture vessel. 2. Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes). Cells that are difficult to detach can be put in 37°C, for several minutes to facilitate detachment. 3. Neutralize Trypsin-EDTA by adding an equal volume of the complete growth media into the culture vessel. 4. Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 125xg for 5 minutes. The actual centrifuge duration and speed may vary depending on the cell type. 5. Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new culture vessels, as desired. 6. Incubate the cells at the recommended conditions.
QC	mRNA and protein expression levels of various markers pre and post differentiation are verified by RT-PCR and western blotting.
Application	Research Use Only.
Storage Condition	Vapor phase of liquid nitrogen, or below -130°C.
BioSafety	II
Disclaimer	1. 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. 2. 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). 3. 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. 4. 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. 5. 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."
Depositor	University of Konstanz

Documents

Supporting Protocol

Other

STR

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 perform extensive downstream characterization, or gene expression profiling of our cell lines. However, you can check the ‘References’ tab to see if a publication is available, or you can contact us to see if RNA or cell lysates are available for your cell line of interest. Please directly contact us for further information at quotes@abmgood.com.

	Does abm's PriGrow series medium contain antibiotics?
	No, the medium does not contain antibiotics and needs to be supplemented by the end-user as desired.

	Do I need to use abm's media and Applied Cell Extracellular Matrix (G422) to culture my cells?
	We strongly encourage using the recommended media and culture conditions described in the "Growth Conditions" section as they have been optimized for cell growth. Other supplier's media and coating conditions have not been tested.

	What is the difference between Applied Cell Extracellular Matrix (G422) and Collagen Coating?
	The main component of our Applied Cell Extracellular Matrix (G422) is type I collagen specifically. For more information on abm's Applied Cell Extracellular Matrix please visit: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html.

	Do I have to use T25 ECM-coated flasks for growing the cells?
	We strongly recommend thawing cells in the T25 flasks specified on the cell-specific product page to ensure optimal post-thaw recovery of the frozen cells before testing other culture vessels.

	If I want to plate these cells to multi-well plates (e.g. 96 well plates) or dishes, how should I prepare the plates?
	For a protocol on how to coat plates and dishes with Applied Cell Extracellular Matrix (Cat. No. G422), please download the “Applied Cell Extracellular Matrix Data Sheet” from here.

	What percentage of Trypsin-EDTA should be used to subculture cells? Can I use Trypsin-EDTA containing phenol red?
	The standard concentration used for subculturing is 0.25% Trypsin-EDTA, available at abm (Cat. No. TM050). Yes, Trypsin-EDTA containing phenol red can be used as it does not interfere with trypsinization.

	How often do I need to change the media?
	Media should be changed every 2-3 days or as specified within the recommended growth conditions.

Why are these cells classified as biosafety level II?

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 desired.

	How long can I store frozen vials?
	Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen vapor phase (or below -130°C) indefinitely without affecting viability.

What is the recommended storage temperature for cells?

In general, if you received:

Live cells: acclimatize for 3-5 hours at the recommended temperature and CO2 conditions stated for the cell line under the Growth Conditions section, and then change media afterwards. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).
Frozen cells: Upon receipt, immediately place cells in liquid nitrogen; or below -130°C.

	Can I substitute heat-inactivated FBS with FBS or vice versa?
	FBS and heat-inactivated FBS are different in their composition; they cannot be substituted for one another.

My cells are not detaching, what method do you recommend to trypsinize the cells?

Gently wash adherent cells with PBS (1X, without calcium or magnesium) to remove trypsin inhibitors before adding the trypsinization agent.
Ensure the correct trypsin concentration is being used (i.e. 0.25%).
Do not use cold trypsin to detach cells as the proteolytic activity will be low. Pre-warm trypsin to 37°C for optimal enzymatic activity.
Incubate the cells with trypsin-edta agent at 37°C to facilitate cell detachment.
Gently tap the side of the culture vessel to facilitate mechanical detachment.

How are live cells shipped?

Live cells are shipped as a live culture in a T12.5 flask. Cells are seeded at an optimal confluency to avoid metabolic waste build up during transport. In addition, we thoroughly test our cell lines for functional freeze-thaw recovery. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).

	Why are cells not attaching and forming clumps after subculturing with trypsin?
	Cells form clumps and display attachment issues when the trypsinization agent is not effectively neutralized. To completely neutralize the trypsin agent, add an equal amount of complete growth media prior to centrifugation. Subtle changes in culture conditions, particularly in pH and the quality of serum used in the growth medium, may also affect the amount of clumping exhibited by the cells.

	Why are my cells forming clumps after plating?
	It is important to ensure cell clumps are broken up through gentle re-suspension by pipetting up and down several times after pelleting cells by centrifugation. This will ensure a homogenous single cell suspension for even plating.

	Do I need to add the selection drug to the complete growth medium?
	We recommend maintaining selection pressure using the drug concentration specified in the Growth Conditions section.

	Where can I find the CoA for my product?
	Certificates of analysis can be found here (https://www.abmgood.com/CoA-Library.html).

References

Scholz, D., Pöltl, D., Genewsky, A., Weng, M., Waldmann, T., Schildknecht, S., & Leist, M. (2011). Rapid, complete and large-scale generation of post- mitotic neurons from the human LUHMES cell line. Journal of Neurochemistry, 119(5), 957–971. https://doi.org/10.1111/j.1471-4159.2011.07255.x

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