Characterization of BCR-ABL Ba/F3 stable clone using Western Blot.
BCR-ABL Cell Line Background
BCR-ABL, breakpoint cluster region – Abelson murine leukemia viral oncogene homolog 1, is an abnormal protein with transforming activity mostly found in chronic myelogenous leukemia (CML) due to the fusion of abnormal configuration of DNA where the ABL gene is fused to the BCR gene (the Philadelphia chromosome); BCR-ABL and its mutants can promote and maintain the malignant behavior of the cancer cells. Ba/F3, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their kinase inhibitors, because some protein kinases can render Ba/F3 cells to be independent of IL-3, while their inhibitors antagonize this effect.
Cell Line Generation
Ba/F3 BCR-ABL cell Line was generated using retrovirus system expressing BCR-ABL 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(BCR-ABL)|
|Cell type:||Pro-B cells|
|Gene ID/Accession #:||N/A|
|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.