KC-5545

U87MG-ERBB2 Cell Line

56660

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Background of U87MG-ERBB2 Cell Line

ERBB2 (Erb-B2 Receptor Tyrosine Kinase 2) is a Protein Coding gene. This gene encodes a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. This protein has no ligand binding domain of its own and therefore cannot bind growth factors. However, it does bind tightly to other ligand-bound EGF receptor family members to form a heterodimer, stabilizing ligand binding and enhancing kinase-mediated activation of downstream signalling pathways, such as those involving mitogen-activated protein kinase and phosphatidylinositol-3 kinase. Allelic variations at amino acid positions 654 and 655 of isoform a (positions 624 and 625 of isoform b) have been reported, with the most common allele, Ile654/Ile655, shown here. Amplification and/or overexpression of this gene has been reported in numerous cancers, including breast and ovarian tumors. Alternative splicing results in several additional transcript variants, some encoding different isoforms and others that have not been fully characterized.

Specifications

Catalog Number	KC-5545
Cell Line Name	U87MG-ERBB2 Cell Line
Clone Number	3#
Host Cell Line	U87MG
Description	U87MG cell line stably expressing exogenous ERBB2 gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 2 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as monolayer
Subculture	Split saturated culture 1:2-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

U87MG-ERBB2 cell line was generated using a lentiviral vector expressing the ERBB2 sequence.

Characterization

Figure 1: Characterization of ERBB2 overexpressing in U87MG-ERBB2 stable clones using FACS.

Figure2: Characterization of U87MG-ERBB2 cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 2 μg/mL Puromycin) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:2-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Van Bockstal M, Lambein K, Denys H, Braems G, Nuyts A, Van den Broecke R, Cocquyt V, De Wever O, Libbrecht L. Histopathological characterization of ductal carcinoma in situ (DCIS) of the breast according to HER2 amplification status and molecular subtype. Virchows Arch. 2014 Sep;465(3):275-89. doi: 10.1007/s00428-014-1609-3. Epub 2014 Jun 29. PMID: 24973889.
Perez EA, Hurvitz SA, Amler LC, Mundt KE, Ng V, Guardino E, Gianni L. Relationship between HER2 expression and efficacy with first-line trastuzumab emtansine compared with trastuzumab plus docetaxel in TDM4450g: a randomized phase II study of patients with previously untreated HER2-positive metastatic breast cancer. Breast Cancer Res. 2014 May 23;16(3):R50. doi: 10.1186/bcr3661. PMID: 24887458; PMCID: PMC4229898.