ROS1 is a receptor tyrosine kinase of the insulin receptor family; the overexpression of overactivity of ROS1 fusion
proteins due to chromosomal rearrangement is associated with various cancers, including glioblastomas and lung cancer. The identification of ROS1 fusion genes as driver genes has broadened the anticancer indication of the variety of the inhibitors of other targets, such as Crizotinib, Alectinib, Ceritinib, and Brigatinib, which can also inhibit the activation of ROS1.
Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.
Specifications
Catalog Number:
KC-3248
Cell Line Name:
Ba/F3 SLC34A2-ROS1-G2032R-F2075V Cell Line
Price:
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Host Cell Line:
Mouse Ba/F3 cell line
Description:
Stable Ba/F3 clone expressing exogenous SLC34A2-ROS1 fusion protein with G2032R F2075V amino acid mutation in ROS1 part.
Quantity:
One vial of frozen cells (5X106 per vial)
Stability:
Stable in culture over a minimum of 10 passages
Application:
Drug screening and biological assays
Freeze Medium:
70% RPMI1640+20% FBS+10% DMSO
Propagation Medium:
RPMI1640+10%FBS
Selection Marker:
Puromycin
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 SLC34A2-ROS1-G2032R-F2075V cell Line was generated using a Lentiviral vector expressing the human SLC34A2-ROS1-G2032R-F2075V fusion sequence.
Characterization using PCR sequencing and WB
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Application
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Cell Resuscitation
1. Prewarm culture medium (RPMI1640 + 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% RPMI-1640 + 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. Gainor, J. F. & Shaw, A. T. Novel targets in non-small cell lung cancer: ROS1 and RET fusions. The Oncologist 18, 865–875 (2013)
2. Bergethon, K. et al. ROS1 rearrangements define a unique molecular class of lung cancers. J. Clin. Oncol. 30, 863–870 (2012).
3. Zou, H. Y. et al. PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations. Proc Natl Acad Sci USA 112, 3493–3498 (2015).
