BT-474 Clone 5

CRL-3247 ^™

BT-474 Clone 5 is an epithelial-like cell line clone that was originally isolated from the mammary gland of a 60-year-old, White female patient with ductal carcinoma. BT-474 (ATCC HTB-20) cells were grown in 10ug/mL Herceptin for several weeks until the resulting cells (clones) were defined as resistant to Herceptin treatment. These resistant cells were grown in standard RPMI with 10% fetal bovine serum for many years under different conditions but the cells enclosed have not been exposed to Herceptin after they were first treated. They were not grown with Herceptin to make sure they stayed resistant. However, these cells have remained resistant for 30+ passages and they still retain the overexpression of HER-2 and are resistant to growth inhibition due to Herceptin.

Product category: Human cells
Organism: Homo sapiens, human
Morphology: epithelial-like
Tissue: Breast; Duct; Mammary gland
Disease: Ductal Carcinoma
Applications: 3D cell culture
Product format: Frozen
Storage conditions: Vapor phase of liquid nitrogen

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Documentation

Product sheet

BSL 1

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.

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.

RPMI-1640 Medium

30-2001

Fetal Bovine Serum (FBS)

30-2020

Dimethylsulfoxide (DMSO)

4-X

General

Specific applications: This cell line is valuable in the study of Her2 positive breast cancer resistant to herceptin therapy

Characteristics

Growth properties: Adherent, patchy
Derivation: BT-474 (ATCC HTB-20) cells were grown in 10ug/mL Herceptin for several weeks until the resulting cells (clones) were defined as resistant to Herceptin treatment. These resistant cells were grown in standard RPMI with 10% fetal bovine serum for many years under different conditions but the cells enclosed have not been exposed to Herceptin after they were first treated. They were not grown with Herceptin to make sure they stayed resistant. However, these cells have remained resistant for 30+ passages and they still retain the overexpression of HER-2 and are resistant to growth inhibition due to Herceptin.
Age: 60 years
Ethnicity: White
Gender: Female
Expression markers: Her2, expressed
Comments: BT-474 (HTB-20) cells were grown in 10 µg/mL Herceptin for several weeks until the resulting cells (clones) were defined as resistant to Herceptin treatment. These resistant cells were grown in standard RPMI with 10% fetal bovine serum for many years under different conditions but the cells enclosed have not been exposed to Herceptin after they were first treated. They were not grown with Herceptin to make sure they stayed resistant. However, these cells have remained resistant for 30+ passages and they still retain the overexpression of HER-2 and are resistant to growth inhibition due to Herceptin. This cell line has overexpressed HER-2 and yet is resistant to herceptin (Trastuzumab) under in vitro conditions but sensitive to ADCC activity.

Handling information

Unpacking and storage instructions

Check all containers for leakage or breakage.
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

The base medium for this cell line is ATCC-formulated RPMI-1640 Medium, ATCC 30-2001. To make the complete growth medium, add the following components to the base medium: fetal bovine serum (ATCC 30-2020) to a final concentration of 10%.

Temperature

37°C

Atmosphere

95% Air, 5% CO₂

Handling procedure

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.

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).
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.
Transfer the vial contents to a centrifuge tube containing 9.0 mL complete growth medium and spin at approximately 125 x g for 5 to7 minutes.
Resuspend cell pellet with the recommended complete growth medium (see the specific batch information for the culture recommended dilution ratio) and dispense into a 25 cm² culture flask. 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).
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 are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Remove and discard culture medium.
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.
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.
Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37°C.

Subcultivation:

Start-up: 6.0 x 10⁴ to 1.0 x 10⁵ viable cells/cm²

Subculture: 6.0 x 10⁴ to 1.5 x 10⁵viable cells/cm²

Medium Renewal: 2 to 3 times per week

Reagents for cryopreservation

RPMI-1640 + 7% FBS + 5% DMSO

Storage temperature: liquid nitrogen vapor phase

Quality control specifications

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

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.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Kute T, et al. Development of herceptin resistance in breast cancer cells. Cytometry A 57(2): 86-93, 2004. PubMed: 14750129

Kute T, et al. Understanding key assay parameters that affect measurements of trastuzumab-mediated ADCC against Her2 positive breast cancer cells. Oncoimmunology 1(6): 810-821, 2012. PubMed: 23162748

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