KC-1508

Ba/F3-TRKB Cell Line

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Background of Ba/F3-TRKB Cell Line

TRKB, also known as NTRK2. This gene encodes a member of the neurotrophic tyrosine receptor kinase (NTRK) family. This kinase is a membrane-bound receptor that, upon neurotrophin binding, phosphorylates itself and members of the MAPK pathway. Signalling through this kinase leads to cell differentiation. Mutations in this gene have been associated with obesity and mood disorders. Alternative splicing results in multiple transcript variants.
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-1508
Cell Line Name	Ba/F3-TRKB Cell Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous TRKB gene.
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+1µg/mL Puromycin+8ng/mL mouse IL-3
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 TRKB cell Line was generated using retrovirus vector expressing TRKB sequence.

Characterization

Figure: Characterization of TRKB overexpressing in Ba/F3 stable clone using FACS.

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% RPMI1640 + 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. Wong J, Higgins M, Halliday G, Garner B. Amyloid beta selectively modulates neuronal TrkB alternative transcript expression with implications for Alzheimer's disease. Neuroscience. 2012 May 17;210:363-74.
2.Zeng F, Zou HQ, Zhou HD, Li J, Wang L, Cao HY, Yi X, Wang X, Liang CR, Wang YR, Zhang AQ, Tan XL, Peng KR, Zhang LL, Gao CY, Xu ZQ, Wen AQ, Lian Y, Zhou XF, Wang YJ. The relationship between single nucleotide polymorphisms of the NTRK2 gene and sporadic Alzheimer's disease in the Chinese Han population. Neurosci Lett. 2013 Aug 29;550:55-9.