hTERT Neonatal Dermal Melanocytes

The base medium for these cells is Dermal Cell Basal Medium (ATCC PCS-200-030). To make the complete medium, add the components of Adult Melanocyte Growth Kit (ATCC PCS-200-042) according to manufacturer instructions.

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 Complete MEL Medium supplemented with 30% 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 30% serum. Do NOT pipette up and down to mix.
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.
5. 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.
6. 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.
7. Pellet the aliquot of cells by centrifugation at approximately 150 x g for 5 to 7 minutes, then remove the cryoprotectant.
8. Resuspend cell pellet into an appropriate medium and use the cell suspension to determine cell viability and concentration.
9. 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 2.0 x 10⁴ and 3.0 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.

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