Characterization of HER2 and its mutants overexpressed in Ba/F3 stable clones using Western Blot.
HER2 Cell Line Background
HER2 is 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 signaling pathways, such as those involving mitogen-activated protein kinase and phosphatidylinositol-3 kinase. 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.
Cell Line Generation
Ba/F3 HER2 Cell Line was generated using retrovirus system expressing HER2 sequence.
Cell Line Characterization
Cell Line Applications
A. Cell-based kinase inhibition screen
B. Cell viability assay
C. In vivo efficacy study
- Harvest and seed the Ba/F3 in 96-well plate (3000 cells/90ul medium).
- Next day, add 10ul 10X serially diluted compound solution each well and incubate the plates for another 72 hours,
- Add 100ul CellTiter-Glo each well, mixed and readout using Envision.
- Plot the dose-responsive curve and fit the IC50 (the concentration of 50% inhibition of DMSO vehicle treated clones) using GraphPad Prism software (Version 5)
Recombinant Cell Line Profile
|Cell line ID:||Ba/F3(HER2)|
|Cell type:||Pro-B cells|
|Gene ID/Accession #:||NM_004448.3|
|Application:||Drug screening and biological assays|
|Morphology:||Mostly single, round (some polymorph) cells in suspension|
|Medium:||RPMI 1640 + 10% FBS|
|Subculture:||Split saturated culture 1:10 every 3 days; seed out at about 1-3 x 105 cells/ml|
|Incubation:||37 °C with 5% CO2|
|Storage:||Frozen in liquid nitrogen with 70% medium, 20% FBS and 10% DMSO|
|Doubling time:||Approximately 20 hours|
Thaw the vial as soon as possible upon receipt to insure its viability.
- Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
- Transfer the vial into biosafety cabinet. Wipe the surface with 70% ethanol and drop the cell suspension gently into a centrifuge tube containing 9.0 mL complete culture medium.
- Spin at ~ 125 x g, for 5- 7 minutes at room temperature.
- Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
- Incubate the culture at 37°, 5% CO2 incubator or refer to the specific culture condition, if possible.