KC-1590

Ba/F3-CD74-ROS1-Cell-Line

22427

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Background of Ba/F3-CD74-ROS1-Cell-Line

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 dependent 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-1590
Cell Line Name	Ba/F3-CD74-ROS1-Cell-Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous CD74-ROS1 fusion protein.
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
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
In Vivo Validation	NA

Cell Line Generation

Ba/F3 CD74-ROS1 cell Line was generated using a Lentiviral vector expressing the human CD74-ROS1 fusion sequence.

Characterization

Figure1: Characterization of ROS1 and its mutants overexpressing in Ba/F3 stable clones using PCR sequencing.

Figure 2: 1. Harvest and seed the Ba/F3 mutant cells in a 96-well plate (3000 cells/90ul medium). 2. Next day, add 10ul 10X serially diluted compound solution to each well and incubate the plates for another 72 hours. 3. Add 100ul Cell Titer-Glo each well, mix and readout using Envision. 4. Plot the dose-responsive curve and fit the IC50 (the centration of 50% inhibition of DMSO vehicle-treated clones) using GraphPad Prism software (Version 5).

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS)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 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

Gainor, J. F. & Shaw, A. T. Novel targets in non-small cell lung cancer: ROS1 and RET fusions. The Oncologist 18,
865–875 (2013)
Bergethon, K. et al. ROS1 rearrangements define a unique molecular class of lung cancers. J. Clin. Oncol. 30,
863–870 (2012).
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).