EML4-ALK Fusion-A549 Isogenic Cell Line Human

The anaplastic lymphoma kinase (ALK) genetic abnormality is a key oncogenic driver, especially in non-small cell lung cancer. The EML4-ALK gene fusion caused by a chromosomal inversion can produce constitutively active ALK tyrosine kinase protein, which leads to enhanced cell survival and cell proliferation. There are multiple EML4-ALK fusion variants, with the most prevalent being variant 1 (E13; A20) in which EML4 intron 13 is fused with ALK intron 20. ATCC isogenic cell line ATCC CCL-185IG was created at ATCC by using CRISPR gene editing technology, and contains EML4-ALK fusion variant 1 (E13; A20). This isogenic cell line (ATCC CCL-185IG)  was created from the A549 parental cell line, ATCC CCL-185, a non-small cell lung cancer cell line. The EML4-ALK fusion in  ATCC CCL-185IG has been intensively validated at the genomic, transcript and protein levels. Furthermore, ATCC CCL-1851G is more sensitive to the ALK inhibitor crizotinib when compared to its parental cell line. ATCC CCL-185IG can be a useful model to study tyrosine kinase signaling pathway, and to screen ALK inhibitors in anti-cancer drug discovery and development.

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.

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.  

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 complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). 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). 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 sheet.

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA Solution (ATCC^® 30-2101) to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
 Cultures can be established between 2 x 10³ and 1 x 10⁴ viable cells/cm². Do not exceed 7 x 10⁴ cels/cm².
6. 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.

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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Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.