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.

It’s important to note that CRL-4064 requires two distinct media formulations:

• Recovery/Thawing medium: Dermal Cell Basal Medium (PCS-200-030) + Adult Melanocyte Growth Kit (PCS-200-042) + 30% FBS
  
  Prepare Recovery/Thawing medium by adding 12 mL FBS (ATCC 30-2020) to 28 mL of the Complete medium  
  
  Note: The above media is stable for 4 weeks at 4 to 8°C.
  
  FBS should not be heat inactivated as this can reduce or destroy serum growth factors, attachment factors and hormones. 
  
  
• Growth medium: Dermal Cell Basal Medium (PCS-200-030) + Adult Melanocyte Growth Kit (PCS-200-042) + 0.5 μg/mL puromycin
  
  Prepare Growth medium by adding 24 uL Gibco Puromycin (10 mg/mL; Thermofisher Scientific catalog # A11138-03 or equivalent) per 480.3 mL Complete medium. 

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. Use 8–10 mL of prepared Recovery/Thawing medium and place it in a T25 flask. Equilibrate the medium in a 37°C, 5% CO₂ incubator for 30 minutes before cell thawing.
2. Thaw the cryovial rapidly in a 37°C water bath, keeping the O-ring and cap dry. Thawing should take no more than 2 minutes.
3. Before the last ice crystal has melted, promptly remove the vial from the water bath. This helps prevent osmotic shock and temperature stress that may compromise cell viability.
4. Immediately disinfect the outside of the vial with 70% ethanol. From this point forward, proceed under strict aseptic conditions
5. Transfer the vial contents into a centrifuge tube containing 1.0 mL of equilibrated thawing medium. Do NOT pipette up and down to mix. Rinse the vial with an additional 1.0 mL of thawing medium and add to the same tube. Do NOT pipette up and down to mix.
6. Centrifuge at approximately 300 x g for 8 minutes, then remove the cryoprotectant. Resuspend cell pellet into Recovery/Thawing medium.
   Incubate the culture at 37°C and 5% CO2
7. After 24 hours, replace medium with Growth medium (complete medium supplemented with 0.5ug/ml puromycin) and incubate the culture at 37°C and 5% CO2

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.

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