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A-549 VIM RFP (ATCC® CCL-185EMT™)

Organism: Homo sapiens, human  /  Cell Type: epithelial  /  Tissue: lung  /  Disease: carcinoma

Permits and Restrictions

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Organism Homo sapiens, human
Tissue lung
Cell Type epithelial
Product Format frozen 1.0 mL
Morphology epithelial-like
Culture Properties adherent
Biosafety Level 2  [Cells containing SV40 and CMV viral sequences]

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 carcinoma
Age 58 years
Gender male
Ethnicity Caucasian
Applications Epithelial to mesenchymal transition (EMT), anti-EMT drug screening, metastatic non small cell lung cancer drug screening, vimentin intermediate filament dynamics.
Storage Conditions liquid nitrogen vapor phase
Images ATCC CCL-185EMT, A-549 VIM RFP Cell Micrograph
Clinical Data 58 years
Caucasian
male
Comments

In lung cancer, Vimentin intermediate filament (IF) proteins have been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-to-mesenchymal transition (EMT/MET), and the metastatic spread of cancer. Vimentin expression is generally upregulated when the cell is in the mesenchymal relative to the epithelial status. Here, we created a Vim-RFP reporter cell line (CCL-185EMT) using the CRISPR/Cas9 gene editing platform and the parental A549 (CCL-185) non-small cell lung cancer (NSCLC) cell line (a gold standard for studying EMT in cancer metastasis within the lung cancer research community). The created CCL-185EMT cell line habors a C-terminal red fluorescent protein (RFP) tag on the vimentin gene. This will enable the tracking of the EMT status of cells in vitro by monitoring RFP expression. The integrity of the Vim RFP knock-in has been verified at the genomic, mRNA and protein level for sequence and expression. Functional evaluation of CCL-185EMT shows sensitivity to anti-EMT drugs PP1 and A83-1. This provides the foundation for the high throughput (HTS) identification of new anti-EMT drugs for metastatic NSCLC therapy. The A549 Vim RFP reporter cell line provides a visualizable, convenient and sensitive platform for research on the mechanisms of metastasis in vitro and the development of new antitumor drugs for metastatic NSCLC.
Complete Growth Medium

For A-549 VIM RFP (ATCC® CCL-185EMT™) cells culture medium should be:

F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) (ATCC® 30-2004™) supplemented with 10% Fetal Bovine Serum (FBS) (ATCC® 30-2020™) and 10 µg/ml blasticidin (Gibco catalog # A11139-03). 
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 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 103 and 1 x 104 viable cells/cm2. Do not exceed 7 x 10 4 cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 6 X 103 and 6 X 104 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial Approximately 2 x 106 cells; Post-thaw Viability ≥ 70%
Volume 1.0 mL
STR Profile
Amelogenin: X,Y
CSF1PO: 10,12
D13S317: 11
D16S539: 11,12
D5S818: 11
D7S820: 8,11
TH01: 8,9.3
TPOX: 8,11
vWA: 14
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 knock-in mutation: PCR- Band at 1894 kb corresponding to Vimentin-RFP junction; Sanger Sequencing – confirms junction sequence
Basic Documentation
Product Sheet
Product Sheet
Certificate of Analysis
Certificate of Analysis
SDS
SDS
Other Documentation
Other Document
Validation Data Other Document
Cell Micrograph
FAQ's
  1. Non-small cell lung cancer (NSCLC) model cell line


    Date Updated: 10/12/2017

  2. CRISPR/Cas9 gene editing technology


    Date Updated: 10/12/2017

  3. Culture conditions for A-549 VIM RFP cells


    Date Updated: 10/12/2017

  4. Validation of VIM RFP expression


    Date Updated: 10/12/2017

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

Gilles C, et al. Vimentin contributes to human mammary epithelial cell migration. J Cell Sci 112 (Pt 24): 4615-4625, 1999. PubMed: 10574710