Cat.No. : CSC-RO0348 Host Cell: Ba/F3
Size: >1x10^6 frozen cells/vial, 1 mL Stability: Stable in culture over a minimum of 10 passages
| Cat. No. | CSC-RO0348 |
| Description | This cell line is engineered to stably overexpress human C-Kit with D816Y mutation. |
| Target Gene | KIT |
| Gene Species | Homo sapiens (Human) |
| Host Cell | Ba/F3 |
| Host Cell Species | Mus musculus (Mouse) |
| Stability | Stable in culture over a minimum of 10 passages |
| Application | Drug screening and biological assays |
| Growth Conditions | 37 °C, 5% CO2 |
| Quality Control | Negative for bacteria, yeast, fungi and mycoplasma. |
| Shipping | Dry ice |
| Storage | Liquid nitrogen |
| Size | >1x10^6 frozen cells/vial, 1 mL |
| Biosafety Level | 2 |
| Thawing & Subculturing Instructions | 1. Thaw cells by gently swirling in a 37°C water bath. To limit contamination, do not submerge the O-ring and cap. 2. When cells are ~70% thawed (~1 min), transfer the vial into a biosafety cabinet, and wipe the surface with 70% ethanol. Allow tube to dry completely. 3. Transfer the cells gently into a 15 mL conical tube containing 10 mL of pre-warmed culture medium (without antibiotic selection marker). Centrifuge cells at ~125 x g for 5~7 min. 4. Remove supernatant without disturbing the pellet, and resuspend cells in 1 mL culture medium (without antibiotic selection marker). Transfer cells to a 6-well plate containing ~2 mL pre-warmed growth medium (without antibiotic selection marker) or a T25 flask containing 5 mL pre-warmed culture medium (without antibiotic selection marker). 5. Incubate the culture at 37°C with 5% CO2. 6. Subculture: split saturated culture 1:4 ~ 1:6 every 3 days; seed out at about 1~3 x 10^5 cells/mL. |
| Growth Properties | Suspension, round |
| Freeze Medium | Frozen with 70% medium, 20% FBS, 10% DMSO |
| Freezing Instructions | Cells are recommended to generate additional frozen stocks at early passages. Frozen stocks should be preserved in a designated cryopreservation medium or in 70% RPMI 1640 + 20% FBS + 10% DMSO (without antibiotic selection marker). 1. Prepare the freezing medium (70% RPMI 1640 + 20% FBS + 10% DMSO, without antibiotic selection marker) 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 x g for 5 minutes at room temperature and carefully aspirate off the medium. 5. Resuspend the cells at a density of at least 3 x10^6 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. |
| Mycoplasma | Negative |
| Format | One frozen vial containing millions of cells |
| Storage | Liquid nitrogen |
| Safety Considerations |
The following safety precautions should be observed. 1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum. 2. No eating, drinking or smoking while handling the stable line. 3. Wash hands after handling the stable line and before leaving the lab. 4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells. 5. All waste should be considered hazardous. 6. Dispose of all liquid waste after each experiment and treat with bleach. |
| Ship | Dry ice |
Activating D816 mutations in the class III receptor tyrosine kinase KIT are associated with the majority of patients with systemic mastocytosis (SM) but are also associated with core binding factor (CBF) AML, making KIT mutations an attractive therapeutic target for the treatment of these cancers. Crenolanib is a potent and selective inhibitor of wild-type and mutant isoforms of the class III receptor tyrosine kinases FLT3 and PDGFRα/β. Notably, crenolanib inhibits a constitutively active mutant FLT3 isoform generated by an aspartate amino acid substitution at codon 835, which is homologous to codon 816 in the KIT gene—indicating sensitivity to the mutant KIT D816 isoform as well. Here, researchers demonstrate that crenolanib targets KIT D816 in SM and CBF AML models: crenolanib inhibits cell proliferation and initiates apoptosis in mastocytosis cell lines expressing these mutations. Targeting specificity was confirmed using syngeneic cell models. The KIT D816 mutation can be targeted with clinically achievable doses of crenolanib. In addition, a rationale is provided for combining crenolanib with cladribine (2-CDA), a standard of care in SM.
The potency of crenolanib in inhibiting cell proliferation and inducing apoptosis in engineered Ba/F3 cell lines (carrying KIT D816V, KIT D816Y, or FLT3 ITD) showed strong correlation with the corresponding tumor cell lines (HMC1.2, p815, or MOLM14, respectively). In contrast, parental IL-3-dependent Ba/F3 cells did not show any significant sensitivity to crenolanib, exhibiting off-target toxicity at 1000 nM (Figures 1A-1F). Notably, comparative analysis of crenolanib versus dasatinib in inducing apoptosis did reveal a higher sensitivity of crenolanib to the mutant KIT D816V target (Figure 1G). This finding suggests that crenolanib could be used to treat mutant KIT malignancies.
Figure 1. Cellular effects of crenolanib are tyrosine kinase-mediated. (Kampa-Schittenhelm K M, et al., 2017)
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Using the Human KIT-D816Y Stable Cell Line - BaF3 has significantly streamlined my research workflow. The cells exhibit robust growth and a stable expression of the D816Y mutation, which saves me precious time that I used to spend on maintaining cell lines.
We have seen a significant improvement in the accuracy of our experiments since switching to the Human KIT-D816Y Stable Cell Line - BaF3. The cells respond predictably to various treatments, which has greatly enhanced the reliability of our drug screening assays.
The customer support and provided documentation for the Human KIT-D816Y Stable Cell Line - BaF3 were outstanding. The comprehensive protocol guide and responsive technical support team helped me set up my experiments quickly and efficiently.
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