KC-3111

Ba/F3-cKIT-F110L-V559D-Y823D Cell Line

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Background of Ba/F3-cKIT-F110L-V559D-Y823D Cell Line

KIT (also known as c-Kit, CD117, and stem cell factor receptor) is a type III receptor tyrosine kinase. Ligand-dependent KIT signaling regulates hematopoietic, mast-cell, melanocyte and germ-cell biology. Activating KIT alterations can confer ligand-independent signaling and are recurrent oncogenic drivers in gastrointestinal stromal tumors and other malignancies.

Specifications

Catalog Number	KC-3111
Cell Line Name	Ba/F3-cKIT-F110L-V559D-Y823D Cell Line
NCBI/UniProt Accession Number	NM_000222.3
Host Cell Line	Ba/F3
Description	A stable Ba/F3 cell line expressing human KIT carrying F110L, V559D, and Y823D substitutions and capable of IL-3-independent growth
Quantity	One vial of frozen cells (≥2×10⁶ cells)
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 at a ratio of 1:10 every 3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Estimated 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3-cKIT-F110L-V559D-Y823D cell line was generated using a retroviral vector expressing the human cKIT-F110L-V559D-Y823D sequence.

Characterization

Figure 1: Characterization of the human cKIT F110L, V559D, and Y823D sequences in Ba/F3-cKIT-F110L-V559D-Y823D stable cells using Sanger sequencing.

Figure 2: Characterization of Ba/F3-cKIT-F110L-V559D-Y823D cell response to cKIT inhibitors following 72-hour treatment using a cell viability assay.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 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 an appropriate culture vessel.
Incubate the cells at 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 complete medium without selective antibiotic.
Subculture the cells at a ratio of 1:10 every 3 days.

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 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

Sartini S, et al. Receptor tyrosine kinase kit and gastrointestinal stromal tumours: an overview. Curr Med Chem. 2011;18(19):2893-2903.
Boikos SA, et al. Gastrointestinal Stromal Tumors: The GIST of Precision Medicine. Trends Cancer. 2018;4(1):74-91.
Isozaki K, Hirota S. Gain-of-Function Mutations of Receptor Tyrosine Kinases in Gastrointestinal Stromal Tumors. Curr Genomics. 2006;7(8):469-475.

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.