KC-1015

Ba/F3-ETV6-NTRK3-G696C-Cell-Line

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Background of Ba/F3-ETV6-NTRK3-G696C-Cell-Line

NTRK2, also named Tropomyosin receptor kinase B (TrkB), is the high affinity catalytic receptor for the neurotrophin and play a major role in neuronal differentiation and survival, the overactivation or overexpression of NTRK2 fusion protein, due to chromosomal rearrangement, was originally found in congenital fibrosarcoma and subsequently found in secretory breast cancer and other type cancer, the identification of NTRK2 fusion genes as driver genes has led to the rapid development of anticancer therapeutics agents, such as LOXO-101, PF-026732240, TSR-011, RXDX-101.

Specifications

Catalog Number	KC-1015
Cell Line Name	Ba/F3-ETV6-NTRK3-G696C-Cell-Line
Host Cell Line	Mouse Ba/F3 cell line
Description	Stable Ba/F3 clone expressing exogenous ETV6-NTRK2 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% RMPI-1640 + 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 ETV6-NTRK2 cell line was generated using retrovirus vector expressing human ETV6-NTRK2 sequence.

Characterization

Figure1: Characterization of ETV6-NTRK2 overexpressing in Ba/F3 stable clones.

Figure 1: 1. Harvest and seed the Ba/F3 cells in 96-well plate (3000 cells/90ul medium). 2. Next day, add 10ul 10X serially diluted compound solution each well and incubate the plates for another 72hours. 3. Add 100ul Cell Titer-Glo each well, mixed 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 (RPMI-1640 supplemented with 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

Vaishnavi, A, A T Le, and R C Doebele. 2015. “TRKing Down an Old Oncogene in a New Era of Targeted Therapy.” Cancer Discovery 5 (1): 25–34.
Jones, David T W, Barbara Hutter, Natalie Jäger, Andrey Korshunov, Marcel Kool, Hans-Jörg Warnatz, Thomas Zichner, et al. 2013. “Recurrent Somatic Alterations of FGFR1 and NTRK2 in Pilocytic Astrocytoma.” Nature Publishing Group 45 (8). Nature Publishing Group: 927–32.