Adipocyte Differentiation Toolkit for Adipose-Derived MSCs and Preadipocytes

1. Check all containers for leakage or breakage.
2. Store the differentiation toolkit at -20°C in a freezer that is not self-defrosting. Do not refreeze the supplements once thawed.
3. If thawed upon arrival, the supplements can be stored at 2 to 8°C as long as they are added to the Adipocyte Basal Medium within 72 hours.
4. Once prepared, both supplemented media are stable for up to three weeks when stored in the dark at 2 to 8°C.

Antimicrobials and phenol red are not required but may be added to the 100 mL bottle of Adipocyte Basal Medium if desired prior to supplementation. The recommended volume of each optional component to be added to the Adipocyte Basal Medium is summarized in Table 1.

1. Follow the instructions for the growth of Adipose-Derived Mesenchymal Stem Cells (ATCC PCS-500-011). It is recommended that the cells not be passaged more than four (4) times before initiating adipocyte differentiation.
2. When cells are 70-80% confluent, passage them into a tissue culture plate at a density of 18,000 cells/cm². Adjust the number of cells and volume of media according to the tissue culture plate used.
3. Example: For a 6 well tissue culture plate with a surface area of 9.5 cm²/well, add a total of 171,000 viable cells to each well containing 2 mL of Mesenchymal Stem Cell Basal Medium (ATCC PCS-500-030) supplemented with Mesenchymal Stem Cell Growth Kit–Low Serum (ATCC PCS-500-040) components.
4. Gently rock the plate back and forth and side to side to evenly distribute cells before incubation. Do not swirl.
5. Incubate the cells at 37°C with 5% CO₂ for 48 hours before initiating adipocyte differentiation.

Adipocyte Differentiation Media Preparation

The adipocyte differentiation process requires two separate media preparations: one for initiation and one for maintenance. Stock solutions of these media can be prepared in tandem in advance as follows: 

1. Thaw all three  components of the differentiation kit and warm to 37°C in a water bath. Note: It may be necessary to shake the AD Supplement and the ADM Supplement upon warming to help re-dissolve any components that may have precipitated out of solution upon freezing.
2. Decontaminate the external surfaces of all three kit components by spraying them with 70% ethanol.
3. Using aseptic technique and working in a laminar flow hood or biosafety cabinet:
   1. Transfer 15 mL of Adipocyte Basal Medium and 1 mL of AD Supplement to a sterile 50 mL conical tube, using a separate sterile pipette for each transfer. This is your working stock of Adipocyte Differentiation Initiation Medium used during the first 96 hours of differentiation.
   2. Add 5 mL of ADM Supplement to the remaining 85 mL of Adipocyte Basal Medium. This is your working stock of Adipocyte Differentiation Maintenance Medium.
4. Tightly cap the each container of media and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
5. Each container of differentiation medium should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, the differentiation media is stable for up to three weeks.

Adipocyte Differentiation Procedure

Initiation Phase

Maintenance Phase

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.