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MCF 10A Ecadherin EmGFP (ATCC® CRL-10317EMT™)

Organism: Homo sapiens, human  /  Tissue: mammary gland; breast  /  Disease: fibrocystic disease

Permits and Restrictions

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Organism Homo sapiens, human
Tissue mammary gland; breast
Product Format frozen 1.0 mL
Morphology epithelial
Culture Properties adherent
Biosafety Level 2

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Disease fibrocystic disease
Age 36 years
Gender female
Ethnicity Caucasian
Applications Epithelial to mesenchymal transition (EMT), anti-EMT drug screening, breast cancer drug screening, E-cadherin expression dynamics.
Storage Conditions liquid nitrogen vapor phase
Comments Cells undergoing EMT often display downregulation of epithelial markers (such as E-cadherin; ECAD) and upregulation of mesenchymal markers (such as vimentin; VIM). Here, we created an ECAD-EmGFP reporter cell line (ATCC CRL-10317EMT) using the CRISPR/Cas9 gene editing platform and the parental MCF 10A (ATCC CRL-10317). The created CRL-10317EMT 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 MCF 10A 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.
Complete Growth Medium The base medium for this cell line (MEBM) along with the additives can be obtained from Lonza/Clonetics Corporation as a kit: MEGM, Kit Catalog No. CC-3150. ATCC does not use the GA-1000 (gentamycin-amphotericin B mix) provided with kit. To make the complete growth medium, you will need to add the following components to the kit (sold separately):
  • 100 ng/ml cholera toxin
Note: Do not filter complete medium
Subculturing
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 medium and rinse monolayer with PBS (ATCC 30-2200) .
  2. Add 3.0 mL of 0.05% trypsin, 0.53 mM EDTA and incubate 37°C for 15 minutes. br />
  3. To neutralize trypsin, add 3 mL solution of 0.1% soybean trypsin inhibitor (ATCC 30-2104). br />
  4. Centrifuge cell suspension at 125 x g for 5 to 10 minutes. Resuspend cell pellet in complete culture medium br />
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
    Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:4 is recommended
    Medium Renewal: Every 2 to 3 days
Cryopreservation
Freeze medium: Complete growth medium supplemented with 7.5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase


Note: Lots manufactured prior to 05/20/2020 may have used a different cryopreservative, contact customer service if needed.
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Cells per Vial Approximately 1 x 106 cells
Volume 1.0 mL
STR Profile
Amelogenin: X
CSF1PO: 10,12
D13S317: 8,9
D16S539: 11,12
D5S818: 10,13
D7S820: 10,11
TH01: 8,9.3
TPOX: 9,11
vWA: 15,17
Sterility Tests Bacteria and yeast: No growth
Mycoplasma: No growth
Viral Testing Hepatitis B: None detected
Cytomegalovirus: None detected
Human immunodeficiency virus: None detected
Epstein-Barr virus: None detected
Human papillomavirus: None detected
Functional Tests Genotype testing for KI mutation: PCR bands at 813 bp and 528 bp corresponding to ECAD-EmGFP LHA and RHA junctions; Sanger sequencing confirms junction sequences.
Name of Depositor ATCC
References

Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7(2): 131-142, 2006. PubMed: 16493418

Richardson F, et al. The evaluation of E-Cadherin and vimentin as biomarkers of clinical outcomes among patients with non-small cell lung cancer treated with erlotinib as second- or third-line therapy. Anticancer Res 32(2): 537-552, 2012. PubMed: 22287743

Soule H, McGrath CM. Immortal human mammary epithelial cell lines. US Patent 5,026,637 dated Jun 25 1991

Pauley RJ, et al. Immortal human mammary epithelial cell sublines. US Patent 5,206,165 dated Apr 27 1993

Soule HD, McGrath CM. A simplified method for passage and long-term growth of human mammary epithelial cells. In Vitro Cell. Dev. Biol. 22: 6-12, 1986. PubMed: 2418007

Soule HD, et al. Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res. 50: 6075-6086, 1990. PubMed: 1975513

Tait L, et al. Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10. Cancer Res. 50: 6087-6094, 1990. PubMed: 1697506

Basic Documentation
Product Sheet
Product Sheet
Certificate of Analysis
Certificate of Analysis
SDS
SDS
Other Documentation
Other Document
Application Data
Restrictions

For commercial accounts, this cell line is only distributed under the terms of a fully signed and executed ATCC® Material Transfer Agreement and Addendum. If the commercial account is screening per completed Addendum, the recipient will be required to pay a Screening Fee (ATCC® ACS-2103F™).

Screening Use is defined as use of Biological Material in small molecule and biologic drug discovery, including initial target identification and validation, assay development, high throughput screening, hit identification, lead optimization, and selection of candidates for clinical development.

If the commercial account is not screening per the completed Addendum, the recipient will not be required to pay a Screening Fee.

In addition to the foregoing, this product's use is governed by the CRISPR Label License Agreement. For information on purchasing a license to use this product for purposes other than those permitted in the CRISPR Label License Agreement, please contact The Broad Institute at partnering@broadinstitute.org.
References

Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7(2): 131-142, 2006. PubMed: 16493418

Richardson F, et al. The evaluation of E-Cadherin and vimentin as biomarkers of clinical outcomes among patients with non-small cell lung cancer treated with erlotinib as second- or third-line therapy. Anticancer Res 32(2): 537-552, 2012. PubMed: 22287743

Soule H, McGrath CM. Immortal human mammary epithelial cell lines. US Patent 5,026,637 dated Jun 25 1991

Pauley RJ, et al. Immortal human mammary epithelial cell sublines. US Patent 5,206,165 dated Apr 27 1993

Soule HD, McGrath CM. A simplified method for passage and long-term growth of human mammary epithelial cells. In Vitro Cell. Dev. Biol. 22: 6-12, 1986. PubMed: 2418007

Soule HD, et al. Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res. 50: 6075-6086, 1990. PubMed: 1975513

Tait L, et al. Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10. Cancer Res. 50: 6087-6094, 1990. PubMed: 1697506