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hTERT-immortalized Dermal Melanocyte

CRL-4059

Product category
Human cells
Product type
hTERT-immortalized cell
Organism
Homo sapiens, human
Tissue
Skin
Disease
Normal
Applications
3D cell culture
Drug development
High-throughput screening
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Price: $5,570.00 ea
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Documentation

Biosafety Icon Biosafety Level 2

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

General

Specific applications
Melanoma, response to UV radiation, psoriasis and other skin diseases, skin trauma (e.g., wound repair, scars, burns), cosmetic research (e.g., skin lightening compounds, skin protecting compounds)

Characteristics

Cells per vial
Approximately 1.5 x 106
Volume
1.0 mL
Growth properties
Adherent
Gender
Female
Karyotype
abnormal, near diploid
Comments
Cells immortalized using the catalytic subunit of the human telomerase (hTERT) gene.

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium

The base medium for this cell is Dermal Cell Basal Medium (485 mL; ATCC PCS-200-030). To make the complete medium add the components of the Melanocyte Growth Kit (ATCC PCS-200-042) and Puromycin at a concentration of 0.5 µg/mL.

The recovery media (without Puromycin) consists of the base medium for this cell line is Dermal Basal Medium (ATCC PCS-200-030), the components of the Melanocyte Growth Kit (ATCC PCS-200-042), and 40% FBS (without puromycin).

Contents of Melanocyte Growth Kit 
  • rh Insulin, 0.5 mL (5 µg/mL final concentration) 
  • Ascorbic Acid, 0.5 mL (50 µg/mL final concentration) 
  • L-Glutamine, 15.0 mL (6 mM final concentration) 
  • Epinephrine, 0.5 mL (1.0 µM final concentration) 
  • Calcium Chloride, 840 µL (1.5 mM final concentration) 
  • Peptide Growth Factor, 1.0 mL 
  • M8 Supplement, 5 mL
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.  

  1. Prepare 8 to 10 ml of MEL Medium supplemented with 20% FBS and without puromycin. Place recovery medium into a T25 flask and equilibrate to temperature and pH for 30 minutes in a 37°C, 5% CO2 incubator prior to thawing cells.
  2. Thaw vial in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
  3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  4. Transfer the vial contents to a centrifuge tube containing  1.0 mL of equilibrated recovery medium, which includes complete medium supplemented with 20% serum. Do NOT pipette up and down to mix.
  5. Rinse the ampoule with 1.0 mL of equilibrated recovery medium and transfer the contents to the same centrifuge tube. Do NOT pipette up and down to mix. Gently mix cells by tapping tube.
  6. From the 3ml of cell suspension, remove an aliquot of cell suspension and transfer to a 1.5ml tube containing an equal amount of equilibrated recovery medium.
  7. Transfer the remaining cell suspension into a T25 tissue culture flask, gently rock to evenly distribute cells, then incubate the culture at 37°C in a suitable incubator.  A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.
  8. Pellet the aliquot of cells by centrifugation at approximately 150 x g for 5 to 7 minutes, then remove the cryoprotectant.
  9. Resuspend cell pellet into an appropriate medium and use the cell suspension to determine ell viability and concentration.
  10. After 24 hours, replace medium with complete medium supplemented with 0.5ug/ml puromycin

         

Subculturing procedure
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with PBS (ATCC 30-2200) to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA for Primary Cells Solution to flask, ensure complete coverage of cells and remove trypsin, then incubate and observe cells until cells have detached (usually within 2 to 3 minutes).
  4. Once detached, add 2.0 to 3.0 mL of a 0.05% soybean trypsin inhibitor and aspirate cells. Transfer cell suspension into a 15ml conical tube.
  5. Add 4.0 to 7.0 mL of complete growth medium to flask to wash and recover residual cells, aspirate cells by gently pipetting. Transfer suspension into previous 15ml conical tube.
  6. Collect cells by centrifugation at 150xg for 5min.
  7. Resuspend cell pellet in 1.5 to 3.0 mL of complete medium. Add appropriate aliquots of the cell suspension to new culture vessels.
    Cultures should be established from cryopreservation between 2 x 104 and 3 x 104 viable cells/cm2.
  8. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 5.0 X 103 and 5.0 X 104 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:10 is recommended
Medium Renewal: 3 times per week
Reagents for cryopreservation
100% Stem Cell Freeze Medium (ATCC ACS-3020)

Quality control specifications

Bacterial and fungal testing
Not detected
Mycoplasma contamination
Not detected
Virus testing
Cytomegalovirus (CMV): Not detected
Human papillomavirus (HPV): Not detected
Human Immunodeficiency virus (HIV): Not detected
Epstein-Barr virus (EBV): Not detected
Hepatitis B virus (HBV): Not detected
Functional tests
Telomerase Activity (TRAP Assay): positive, ≥ 4 repeats; ICC Staining: posititve for Trp-1 (MEL-5); negative for TE-7
Population doubling time
Approximately 46 hrs
STR profiling
Amelogenin: X
CSF1PO: 12
D13S317: 12,13
D16S539: 11
D5S818: 11,12
D7S820: 8,10
THO1: 6,7
TPOX: 8,11
vWA: 15,17

History

Depositors
ATCC

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Material Transfer Agreement Addendum for Screening Applications

For-profit organizations
For every order of this item, you must provide a signed Material Transfer Agreement Addendum for Screening Applications. We cannot ship this item until we receive this addendum. The person signing the addendum as the principal investigator must match the end user as listed on the applicable sales order for the item.

Email the signed addendum to [email protected] with a reference to both your account and sales order numbers. Once received, your addendum will be reviewed, and this item will be released for shipment if all requirements are met. Additional fees may apply if this product is being used for a screening use (ATCC ACS-2103F), and these fees will be applied after your order is confirmed. If you need assistance with your order, please contact our Customer Care team or your applicable distributor.

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Frequently Asked Questions

Need assistance with this product? Contact our Technical Support team.

Telephone

Telephone

US and Puerto Rico

800-638-6597

Outside the US

+1-703-365-2700
hours

Hours of Operation

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