KC-5395

Ba/F3-FLT3-ITD-F691L-Luc2 Cell Line

65459

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Background of Ba/F3-FLT3-ITD-F691L-Luc2 Cell Line

FLT3 (Fms Related Receptor Tyrosine Kinase 3) is a Protein Coding gene. FLT3 encodes a receptor tyrosine kinase that regulates hematopoiesis in hematopoietic progenitor cells. It is a class III receptor activated by FLT3 ligand binding, which induces homodimer formation and autophosphorylation at the plasma membrane. This activation supports proliferation, survival, and differentiation of hematopoietic cells and dendritic cells. Diseases associated with FLT3 include Leukemia, Acute Myeloid and Acute Myeloid Leukemia With Minimal Differentiation.

Specifications

Catalog Number	KC-5395
Cell Line Name	Ba/F3-FLT3-ITD-F691L-Luc2 Cell Line
NCBI/UniProt Accession Number	NM_004119.3
Clone Number	1#
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous FLT3 bearing ITD-F691L 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	NA
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 3 days
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-F691L-Luc2 cell line was generated using a lentiviral vector expressing the FLT3-ITD-F691L-Luc2 sequence.

Characterization

Figure 1: Characterization of the Ba/F3-FLT3-ITD-F691L-Luc2 cell line stable clone using Bright-Lite Luciferase Assay System in the conditions of different cell number.

Figure 2: Characterization of FLT3-ITD-F691L mutation in the Ba/F3 stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (RPMI1640+10%FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:10 every 3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

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. 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.
3. Negotei C, Colita A, Mitu I, Lupu AR, Lapadat ME, Popovici CE, Crainicu M, Stanca O, Berbec NM. A Review of FLT3 Kinase Inhibitors in AML. J Clin Med. 2023 Oct 10;12(20):6429. doi: 10.3390/jcm12206429. PMID: 37892567; PMCID: PMC10607239.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.