LUHMES

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.

• 1% N2 supplement (Gibco-Invitrogen Cat No 17502-048)
• 40 ng/ml b-FGF ( basic recombinant human Fibroblast Growth Factor; Gibco-Invitrogen Cat No 13256-029) added fresh at the last moment

To insure 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.

Note: Culture flasks should be pre-coated with 50µg/mL poly-L-ornithine and then with 1µg/mL human fibronectin. (See Subculturing Procedure)

1. Thaw the vial by gentle agitation 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).
2. 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.
3. Transfer the vial contents to a centrifuge tube containing 9.0 mL culture medium without bFGF and spin at approximately 125 x g for 5 to 10 minutes.
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio) and dispense into a pre-coated vented 75 cm² culture flask.  It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. 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.

These cells should be recovered from cryopreservation and subcultured only on culture flasks that are sequentially pre-coated with 50 µg/mL poly-L-ornithine (Sigma, Cat. No. P-3655 or equivalent) and then with 1 µg/mL Human Fibronectin (Sigma, Cat. No. F-0895 or equivalent).

1. Add 7.0 mL freshly diluted 50 µg/mL poly-L-ornithine to T-75 cm² flask and allow flask to sit over night at room temperature.
2. Remove and discard poly-L-ornithine solution. Rinse flask 3 times with culture grade water (Hyclone cat#SH30529.01). Discard last rinse and allow flask to air-dry uncapped and standing upright in a biological cabinet.
3. Add 5.0 mL freshly diluted 1 µg/mL fibronectin and incubate 3 hours at 37°C.
4. Remove and discard fibronectin solution. Rinse flask 3 times with culture grade water (Hyclone cat#SH30529.01). Discard last rinse and allow flask to air-dry uncapped and standing upright in a biological cabinet.
5. Flask is ready for use when dry.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 10.0 mL Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS).
3. Add 4.0 mL of freshly diluted, pre-warmed 0.025% Trypsin-0.1 g/L EDTA solution (see formula below) to flask and incubate for 3 minutes at 37°C. Knock the flask several times against the palm of your hand to dislodge cells. Observe flask under an inverted microscope to be sure cells have come off.
4. Add 6.0 mL of pre-warmed wash medium (see Note above) and aspirate cells by gently pipetting.
5. Transfer cell suspension to a centrifuge tube and spin at approximately 190 x g for 7 minutes. Discard supernatant and knock the tube against the palm of your hand to loosen the cell pellet. Using a 1 mL pipet, add 1.0 mL complete growth medium and pipet the pellet up and down to resuspend the cells (avoid creating foam or bubbles).
6. Add an additional 2.0 mL of complete growth medium and dissociate cells further by pipetting up and down.
7. Adjust cell concentration by adding the necessary volume of complete growth medium needed to seed new flasks. Pipet up and down to evenly resuspend cells.
8. Add appropriate aliquots of the cell suspension to new pre-coated vented culture flasks.
9. Incubate cultures in 5% CO₂/95% air at 37°C.

2X Trypsin-EDTA Solution: 2X Trypsin-EDTA solution is 0.05% Trypsin-0.2g/L EDTA. Before use this must be diluted 1:1 in Ca++/Mg++ free Dulbucco's phosphate-buffered saline (D-PBS) to 0.025% Trypsin-0.1g/l EDTA
To make 1 liter of 2X Trypsin-EDTA solution:

1. Add the following to 500 ml ddH₂O:
   1. 8 g NaCl
   2. 0.4 g KCL
   3. 0.58 g NaHCO₃
   4. 1 g Dextrose
   5. 0.2 g EDTA
   6. 0.5 g Trypsin (Sigma, Cat. No. T7409)
2. Bring volume up to 1 liter with ddH₂O, and pH to 7.4 with HCL.
3. Incubate at 37°C for at least 1 hour to activate the trypsin.
4. Sterile filter (0.2 µm) and make aliquots.
5. Refrigerate at 4°C overnight and then store at -20°C.
6. Before use, dilute trypsin 1:1 with Ca++/Mg++ free Dulbucco?s phosphate-buffered saline (D-PBS) and warm to 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.