HFF-1

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. 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. 
2. Remove the vial from the water bath as soon as the contents are half way thawed (approximately 90 seconds), 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’s contents plus 5 mL of complete growth medium to a 15 mL centrifuge tube. Use an additional 1 mL of media to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 mL of complete growth media to bring the total volume to 10 mL.
4. Gently mix and pellet the cells by centrifugation @ 270 xg for 5 minutes.
5. Discard the supernatant and resuspend the cells with 10 mL fresh growth medium (warm) and plate the cells at seed density of 0.8 X10^{4 c}ells/cm².
6. Add more fresh growth medium (warm) to obtain the total volume recommended for the flask.
7. Incubate 37^°C in a 5%CO₂ in air atmosphere.

Fluid change twice a week or when pH decreases. 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 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).

Procedure:
To insure the highest level of viability, be sure to warm media and Trypsin/ EDTA to 37ºC before using it on the cells.

Cells should be split when they reach confluency. A split ratio of 1:5 to 1:7 is recommended. Volumes used in this protocol are for 225 cm² (T225); proportionally reduce or increase amount of dissociation medium for culture flasks of other sizes.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 1X PBS (SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
3. Add 5 mL of Trypsin-EDTA (0.25% (w/v) Trypsin-0.53 mM EDTA solution, ATCC# 30-2101) solution to flask and incubate for 1 minute, gently tapping the flask observe cells under an inverted microscope until cells detach (usually within 1 to 2 minutes).
4. Add 6.0 to 8.0 mL of complete growth medium and rinse surface of the flask to detach all cells. Gently pipetting up and down will break cell clumps.
5. Transfer all cells into a centrifuge bottle or tube and centrifuge at 270 xg for 5 minutes.
6. Remove and discard the supernatant
7. Add 10 mL complete growth medium to cell pellet and with 10 mL pipette resuspend the cells gently (create a single-cell suspension).
8. Add more complete growth medium to cell suspension as needed to plate cells at approximately 5x10⁶/T225 flask.
9. Place flasks in incubator @ 37°C with a 5% CO₂ in air atmosphere.

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 5th edition, published by Alan R. Liss, N.Y., 1994.  

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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