KC-0981

Ba/F3 FLT3-ITD-D835V Cell Line

54319

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Background of Ba/F3 FLT3-ITD-D835V Cell Line

Fms like tyrosine Kinase 3 (FLT3), also named as CD135, FLK2, is receptor kinase receptor, expressed on the surface of many hematopoietic progenitor cells. Overactivation of FLT3 due to mutation and overexpression can lead to the development and poor prognosis of Leukemia, the identification of FLT3 as a driver gene has led to the repaid development of anti-AML therapeutics, including Sunitinib, Sorafenib and Quizartinib (AC220).
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.
D835 residue mutation is a secondary kinase domain mutation in FLT3-ITD, typically associated with acquired clinical resistance to effective FLT3 tyrosine kinase inhibitors (TKIs). Molecular docking studies have suggested that D835 mutations primarily confer resistance by stabilizing an active Asp-Phe-Gly in (‘DFG-in’) kinase conformation unfavorable to the binding of type II FLT3 TKIs, which target a ‘DFG-out’ inactive conformation.

Specifications

Catalog Number	KC-0981
Cell Line Name	Ba/F3 FLT3-ITD-D835V Cell Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous FLT3 bearing ITD duplicate and D835V amino acid double mutations
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

Cell Line Generation

Ba/F3-FLT3-ITD-D835V-cell-Line was generated using retrovirus vector expressing human FLT3-ITD-D835V sequence.

Characterization

Figure 1: Characterization of FLT3 overexpression in the Ba/F3 stable clone using FACS.

Figure 2.:Characterization of dose-response curves for FLT3 inhibitors on Ba/F3-FLT3-ITD-D835V cells.

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% 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.

References

1. Quentmeier, H., Reinhardt, J., Zaborski, M. & Drexler, H. G. FLT3 mutations in acute myeloid leukemia cell lines. Leukemia 17, 120–124 (2003).
2. Smith, C. C. et al. Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature 485, 260–263 (2012).
3. Moore, A. S. et al. Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns. Leukemia 26, 1462–1470 (2012).
4.Wang Z, Hu B, An Y, Wang J. Exploring the Resistance Mechanisms of Distal D835V Mutation in FLT3 to Inhibitors. Oxid Med Cell Longev. 2022 Mar 28;2022:3720026. doi: 10.1155/2022/3720026. PMID: 35387260; PMCID: PMC8979743.