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A-549 VIM RFP

CCL-185EMT

Product category
Human cells
Product type
Reporter-labeled cell
Cell model
Organism
Homo sapiens
Cell type
epithelial cell
Morphology
epithelial-like
Tissue
Lung
Disease
Carcinoma
Applications
3D cell culture
Assay development
Cancer research
Drug development
High-throughput screening
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Price: $4,586.00 ea
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Documentation

Biosafety Icon BSL 2

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Cells contain cytomegalovirus (CMV) DNA sequences

Cells contain SV40 sequences

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

F-12K Medium (Kaighn's Modification of Ham's F-12 Medium)

30-2004

Price: $20.00 ea

Fetal Bovine Serum (FBS)

30-2020

Price: $645.00 ea

Trypsin-EDTA Solution, 1X

30-2101

Price: $14.00 ea

Dimethylsulfoxide (DMSO)

4-X

Price: $49.00 ea

Detailed product information

General

Specific applications
Epithelial to mesenchymal transition (EMT), anti-EMT drug screening, metastatic non small cell lung cancer drug screening, vimentin intermediate filament dynamics.

Characteristics

Cells per vial
Approximately 2.0 x 106
Volume
1.0 mL
Growth properties
Adherent
Age
58 years
Ethnicity
White
Gender
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.

Handling information

Unpacking and storage instructions
  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.
Complete 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). 


Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure

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. Initial seeding density is 1 x 104 and 2 x 104 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.

         

Subculturing procedure
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

Quality control specifications

Bacterial and fungal testing
Not detected
Mycoplasma contamination
Not detected
Virus testing
Cytomegalovirus (CMV): Not detected
Human papillomavirus (HPV): Not detected
Human Immunodeficiency virus (HIV): Not detected
Epstein-Barr virus (EBV): Not detected
Hepatitis B virus (HBV): Not detected
Functional tests
Genotype Testing for knock-in mutation: PCR- Band at 1894 kb corresponding to Vimentin-RFP junction; Sanger Sequencing – confirms junction sequence
STR profiling
Amelogenin: X,Y
CSF1PO: 10,12
D13S317: 11
D16S539: 11,12
D5S818: 11
D7S820: 8,11
THO1: 8,9.3
TPOX: 8,11
vWA: 14
Viability
≥ 70%

Legal disclaimers

Intended use
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.
Warranty

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.

Disclaimers

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.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

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

Permits & Restrictions

Material Transfer Agreement Addendum for Screening Applications

For-profit organizations
For every order of this item, you must provide a signed Material Transfer Agreement Addendum for Screening Applications. We cannot ship this item until we receive this addendum. The person signing the addendum as the principal investigator must match the end user as listed on the applicable sales order for the item.

Email the signed addendum to [email protected] with a reference to both your account and sales order numbers. Once received, your addendum will be reviewed, and this item will be released for shipment if all requirements are met. Additional fees may apply if this product is being used for a screening use (ATCC ACS-2103F), and these fees will be applied after your order is confirmed. If you need assistance with your order, please contact our Customer Care team or your applicable distributor.

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

This material is subject to the following restrictions in addition to those outlined in the ATCC Material Transfer Agreement:

  1. CRISPR Label License 

For information on obtaining additional rights, please contact:

ATCC Licensing
Email: [email protected]

This material is subject to the following restrictions in addition to those outlined in the ATCC Material Transfer Agreement:

  1. CRISPR Label License, ERS Genomics 

For information on obtaining additional rights, please contact:

ATCC Licensing
Email: [email protected]

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

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

Frequently Asked Questions

Need assistance with this product? Contact our Technical Support team.

Telephone

Telephone

US and Puerto Rico
800-638-6597

Outside the US
+1-703-365-2700
hours

Hours of Operation

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