KC-1306

CHOK1-FLT3 Cell Line

21895

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Background of CHOK1-FLT3 Cell Line

The FLT3 gene encodes a Class III receptor tyrosine kinase that regulates hematopoietic functionThe receptor is activated by binding of the fms associated tyrosine kinase 3 ligand to the extracellular domain, which induces the formation of homodimers in the plasma membrane, resulting in the autophosphorylation of the receptor. Activated receptor kinases then phosphorylate and activate multiple cytoplasmic effector molecules in pathways involved in apoptosis, proliferation, and differentiation of bone marrow hematopoietic cells.

Specifications

Catalog Number	KC-1306
Cell Line Name	CHOK1-FLT3 Cell Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous FLT3 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 + 5μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 every 2-3 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CHOK1 FLT3 Cell Line was generated using a lentiviral vector expressing the FLT3 sequence.

Characterization

Figure 1: Characterization of FLT3 overexpression in the CHOK1 FLT3 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (F12K supplemented with 10% FBS and 5μg/mL Puromycin)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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% F12K + 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. Daver N, Schlenk RF, Russell NH, Levis MJ. Targeting FLT3 mutations in AML: review of current knowledge and evidence. Leukemia. 2019 Feb;33(2):299-312. doi: 10.1038/s41375-018-0357-9. Epub 2019 Jan 16. PMID: 30651634; PMCID: PMC6365380.
2. Kiyoi H, Kawashima N, Ishikawa Y. FLT3 mutations in acute myeloid leukemia: Therapeutic paradigm beyond inhibitor development. Cancer Sci. 2020 Feb;111(2):312-322. doi: 10.1111/cas.14274. Epub 2019 Dec 30. PMID: 31821677; PMCID: PMC7004512.
3. Fedorov K, Maiti A, Konopleva M. Targeting FLT3 Mutation in Acute Myeloid Leukemia: Current Strategies and Future Directions. Cancers (Basel). 2023 Apr 15;15(8):2312. doi: 10.3390/cancers15082312. PMID: 37190240; PMCID: PMC10136888.
4. Arai Y, Chi S, Minami Y, Yanada M. FLT3-targeted treatment for acute myeloid leukemia. Int J Hematol. 2022 Sep;116(3):351-363. doi:10.1007/s12185-022-03374-0. Epub 2022 May 9. PMID: 35532877.