KC-4945

Ba/F3-ERBB2-S310F-ERBB3 Cell Line

59806

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Background of Ba/F3-ERBB2-S310F-ERBB3 Cell Line

ERBB3, also known as HER3, is a transmembrane protein belonging to epidermal growth factor receptor (ERBB) family of receptor tyrosine kinase. The kinase domain impaired ERBB3 can from active heterodimer with other member of ERBB family, this heterodimer, mostly with ERBB2 after binding with its ligand NRG1 or NRG2, play an important role in cancer growth, invasion, metastasis and chemotherapeutic resistant.
ERBB2, also known as CD340. 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-4945
Cell Line Name	Ba/F3-ERBB2-S310F-ERBB3 Cell Line
NCBI/UniProt Accession Number	NM_004448 NM_001982
Clone Number	2-6#
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous ERBB2 gene bearing S310F mutations and ERBB3 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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640 + 10%FBS + 1μg/mL Puromycin + 1000μg/mL G418
Selection Marker	Puromycin \| G418
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 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 20 hours
Mycoplasma Status	Negative

Cell Line Generation

Ba/F3 ERBB2 S310F ERBB3 Cell Line was generated using retrovirus vector expressing ERBB2 S310F ERBB3 sequence.

Characterization

Figure 1: Characterization of ERBB2 S310F ERBB3 mutation in the Ba/F3 stable clone using PCR sequencing.

Figure 2: Characterization of human ERBB2 S310F ERBB3 overexpression in the Ba/F3 ERBB2 S310F ERBB3 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Baselga, J. & Swain, S. M. Novel anticancer targets: Revisiting ERBB2 and discovering ERBB3. Nat. Rev. Cancer 9, 463ÿ475 (2009).
2. Lee SY, Yoo TK, Kim J, Chung IY, Ko BS, Kim HJ, Lee JW, Son BH, Lee SB. Clinical significance of microinvasive breast cancer across the different subtypes and human epidermal growth factor receptor 2 expression levels. Breast Cancer Res Treat. 2023 Jul;200(1):47-61.