hTERT Immortalized RPTEC Growth Kit

Unpacking and Storage Instructions

1. Check all containers for leakage or breakage.
2. Store the growth kit(s) at either -20°C in a freezer that is not self-defrosting or at -70°C for long term storage. If thawed upon arrival, the Growth Kit can be stored at 2°C to 8°C and added to the basal medium within 72 hours of receipt.

Preparation of Complete Growth Media

1. Obtain one hTERT immortalized RPTEC Growth Kit from the freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit and mix by gentle vortex or inversion just prior to adding them to the basal medium. Note: Only use 8.0 mL of Supplement B to obtain the optimal final concentration.
3. Obtain one bottle of DMEM/F12 Medium (500 mL) from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the indicated volume of each growth kit component, as indicated in Table 1, to the bottle of basal medium using a separate sterile pipette for each transfer.

Table 1. hTERT immortalized RPTEC Growth Kit

The final concentration for each growth kit component in the complete hTERT immortalized RPTEC growth medium is as follows:

25 ng/mL Hydrocortisone

3.5 µg/mL Ascorbic Acid

8.65 ng/mL Sodium selenite

5.0 µg/mL Transferrin

5.0 µg/mL insulin

5 pM Triiodo-L-thyronine

25 ng/mL  Prostaglandin E1

10 ng/mL rhEGF

1.2 mg/mL Sodium Bicarbonate

Required but not supplied: G418 solution MUST be added to the above medium to a final concentration of 0.1 mg/mL G418 to maintain the selective pressure for immortalization.

Fetal Bovine Serum is not required for proliferation but may be added if it is desired to transfect the cells. The recommended volume of each optional component to be added to the complete growth media is summarized in Table 2.

Table 2. Addition of Serum  (Optional)

6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete growth media is stable for four weeks.

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.

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