FGFR3 is a member of the fibroblast growth factor receptor family which play a role in mitogenesis and differentiation. The FGFR3 gene produces various forms of the FGFR3 protein; the location varies depending on the isoform of the FGFR3 protein. Since the different forms are found within different tissues the protein is responsible for multiple growth factor interactions. Gain of function mutations in FGFR3 inhibits chondrocyte proliferation and underlies achondroplasia and hypochondroplasia.
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.
Mostly single, round (some polymorph) cells in suspension
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 20 hours
Cell Line Generation
Ba/F3-FGFR3-TACC3-L608V cell line was generated using a lentiviral vector expressing the human FGFR3-TACC3 sequence bearing L608V mutation.
Characterization using PCR sequencing and WB
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.
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
1. "FGFR3 gene". Genetics Home Reference. U.S. National Library of Medicine. Retrieved 2018-09-27.
2. Wang Y, Liu Z, Liu Z, Zhao H, Zhou X, Cui Y, Han J (May 2013). "Advances in research on and diagnosis and treatment of achondroplasia in China". Intractable & Rare Diseases Research. 2 (2): 45–50. doi:10.5582/irdr.2013.v2.2.45
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