BT-474 ECAD-EmGFP

Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the incidence of cancer metastasis and drug resistance, their impact in cancer progression and patient survival is not fully understood (Nieto et al. 2016). During EMT, epithelial cells lose their polarity, as well as their cell-cell adhesions, and acquire the motile and invasive characteristics of mesenchymal cells (Hay 1995). Proteins such as vimentin intermediate filament (IF) proteins are generally upregulated when the cell is in the mesenchymal relative to the epithelial status (Gilles et al. 1999; Thiery and Sleeman 2006; Richardson et al. 2012; Lamouille et al. 2014). Here, we created an ECAD-EmGFP reporter cell line (HTB-20EMT) using the CRISPR/Cas9 gene editing platform and the parental BT-474 breast ductal carcinoma cell line (ATCC HTB-20). The HTB-20EMT cell line harbors a C-terminal green fluorescent protein (EmGFP) tag on the E-cadherin gene. This will enable the tracking of the EMT status of cells in vitro by monitoring GFP expression. The integrity of the ECAD-EmGFP knock-in has been verified at the genomic, mRNA and protein level for sequence and expression. The BT-474 ECAD-EmGFP reporter cell line provides a convenient and sensitive platform for research on the mechanisms of metastasis in vitro and the development of new antitumor drugs for metastatic breast cancer.

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.

The base medium for this cell line is Dulbecco's Modified Eagle's Medium (DMEM; ATCC 30-2002). To make the complete medium add the following component:

• 10% Fetal Bovine Serum (FBS; ATCC 30-2020)

To ensure 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 250 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.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution 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 10⁴ and 2 x 10⁵ viable cells/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.

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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.