Cat.No. : CSC-RO0349 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-RO0349 |
| Description | This cell line is engineered to stably overexpress human C-Kit with D816H 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 |
Avapritinib, the sole approved inhibitor, targets D842V-mutant gastrointestinal stromal tumors (GIST), the primary mutation of PDGFRA. Researchers utilize the Human KIT-D816H Stable Cell Line to investigate the mechanism of action of avapritinib, an approved inhibitor for D842V-mutant GIST. Despite its efficacy, patients develop resistance mutations or neuro-cognitive side effects, necessitating further drug development. By elucidating avapritinib's binding mode through crystal structures of wild-type and mutant PDGFRA and KIT, researchers identify a sub-pocket (Gα-pocket). This information aids in designing avapritinib derivatives to overcome drug resistance and limit blood-brain barrier penetration, facilitating the development of next-generation therapeutics.
Figure 1. The impact of chemical modifications on the structure of avapritinib to overcome resistance mutations was investigated by researchers. Alterations were made to key elements like the Gα-pocket and linker lengths to explore potential modifications for enhancing efficacy and reducing blood-brain barrier penetration. Synthetic compounds were evaluated against mutant forms of KIT and PDGFRA using biochemical and cellular assays, providing insights into structure-activity relationships and off-target effects. (Teuber A, et al., 2024)
Utilizing Creative Biogene's Human KIT-D816H Stable Cell Line - BaF3 provides an effective means to replicate tumor-associated genetic mutations, particularly the KIT gene D816H mutation, offering researchers a stable and dependable experimental platform. The use of this cell line aids researchers in gaining a deeper understanding of tumorigenesis mechanisms, exploring potential therapeutic strategies, and advancing research in the field of oncology.
A: Ba/F3 cells were likely selected for their cytokine-dependent growth and suitability for studying oncogenic kinase activity and drug sensitivity associated with EGFR mutations, such as the D770del_ins_GY mutation.
A: Stability was likely confirmed through methods such as immunoblotting, functional assays measuring downstream signaling, or cell viability assays in the absence of growth factors, with continuous selection pressure applied.
A: Characterization may involve analysis of EGFR phosphorylation, downstream signaling pathways, and functional implications in cell proliferation, survival, and response to EGFR inhibitors such as afatinib or dacomitinib.
A: Quality control likely included confirmation of EGFR-D770del_ins_GY expression levels, validation of its kinase activity and drug sensitivity, assessment of off-target effects, and validation of phenotypic changes associated with EGFR modulation.
A: Comparative analysis with patient-derived samples or in vivo models helps validate the relevance of EGFR-D770del_ins_GY expression in cancer progression, metastasis, and response to EGFR-targeted therapies, guiding the development of personalized treatment strategies for EGFR-mutant cancers, particularly those harboring uncommon mutations like D770del_ins_GY.
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Exceptional stability! The Human KIT-D816H Stable Cell Line in Ba/F3 cells ensures consistent expression of the KIT D816H mutation, providing reliable results in my research on leukemia and targeted therapy.
Enabling advanced studies! With stable KIT-D816H expression, I can investigate drug resistance mechanisms and novel therapeutic strategies with confidence, advancing our understanding of leukemia biology.
Impressive reliability! This cell line surpasses expectations, serving as a valuable tool for studying KIT-D816H-targeted therapies and personalized treatment approaches for KIT-mutant cancers.
Optimizing research workflows! Its stable expression streamlines experimental procedures, facilitating efficient data collection and analysis, and accelerating discoveries in drug resistance mechanisms and leukemia pathogenesis.
An invaluable resource! The Human KIT-D816H Stable Cell Line has significantly enhanced my research capabilities, offering valuable insights into KIT-driven oncogenesis and potential therapeutic strategies for leukemia treatment.
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