hTERT-immortalized Dermal Melanocyte

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.

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).

• 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

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 40% 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 40% serum. Do NOT pipette up and down to mix.
5. 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.
6. After 24 hours, replace medium with complete medium supplemented with 0.5ug/ml puromycin.

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.

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.

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.