Cat.No. : CSC-RO0333 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-RO0333 |
| Description | This cell line is engineered to stably overexpress exogenous EML4-ALK fusion gene with L1198F mutation in the ALK part. |
| Target Gene | EML4-ALK |
| 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 |
The rearrangement of the anaplastic lymphoma kinase (ALK) gene occurs in about 3%-5% of non-small cell lung cancer (NSCLC) patients, making it a critical target for treatment with ALK inhibitors (TKIs). Over time, several generations of TKIs have been developed, with second-generation inhibitors like ceritinib showing great success in treating ALK-positive NSCLC. However, resistance often emerges, driven by secondary mutations like ALK-L1198F. In this study, researchers explored how this mutation confers resistance to a variety of ALK inhibitors, including ceritinib, while paradoxically retaining sensitivity to crizotinib, a first-generation TKI. Through experiments with patient-derived cells and Ba/F3 cells expressing the ALK-L1198F mutation, they found that this mutation increased the cells' resistance to next-generation inhibitors like lorlatinib and brigatinib, but lowered resistance to crizotinib, suggesting the mutation alters drug-binding properties.
Figure 1. The researchers used Ba/F3 cells expressing ALK-L1256F or I1171N + L1256F mutations to investigate drug resistance mechanisms. They treated these cells with various ALK inhibitors to evaluate phospho-ALK suppression and calculate IC50 values, aiming to understand the differential drug sensitivity, particularly lorlatinib resistance and alectinib sensitivity. (Okada K, et al., 2019)
Creative Biogene offers a Human EML4-ALK-L1198F Stable Cell Line, which can be used in similar research to study drug resistance mechanisms in NSCLC. This cell line provides a powerful tool for evaluating ALK inhibitors and understanding resistance patterns, aiding the development of new targeted therapies.
A: The Human EML4-ALK-L1198F Stable Cell Line - BaF3 is a valuable tool for investigating resistance mechanisms in ALK-positive lung cancer. The stable expression of the EML4-ALK-L1198F fusion protein in BaF3 cells allows researchers to study how specific mutations, such as L1198F, confer resistance to ALK inhibitors. This model enables the screening of novel therapeutic strategies targeting resistant variants and the identification of potential combination therapies to overcome resistance.
A: When culturing the Human EML4-ALK-L1198F Stable Cell Line - BaF3, it's crucial to maintain optimal growth conditions, such as the correct temperature (37°C), CO2 levels (5%), and a sterile environment to prevent contamination. Regular genotyping is recommended to confirm the stability of the EML4-ALK-L1198F mutation. Additionally, using a medium supplemented with the appropriate growth factors and antibiotics can help maintain selective pressure, ensuring the continued presence of the desired genetic alteration.
A: The L1198F mutation in the ALK domain of the Human EML4-ALK-L1198F Stable Cell Line - BaF3 confers a unique profile of sensitivity to ALK inhibitors. This mutation can result in either increased resistance or sensitivity to different ALK inhibitors, depending on the specific compound. For instance, some ALK inhibitors that are effective against wild-type ALK might be less effective against cells harboring the L1198F mutation, highlighting the importance of using this cell line for testing the efficacy of novel ALK inhibitors.
A: The BaF3 cell line background provides a robust platform for the Human EML4-ALK-L1198F Stable Cell Line, as BaF3 cells are IL-3 dependent for growth. This dependency allows for the assessment of cell viability and proliferation in the absence of IL-3 when the EML4-ALK fusion protein is expressed, serving as a model for oncogenic signaling. Furthermore, the BaF3 system is well-characterized and widely used in drug discovery, making it an ideal background for evaluating the efficacy and specificity of ALK inhibitors in a cellular context relevant to ALK-positive cancers.
A: Yes, the Human EML4-ALK-L1198F Stable Cell Line - BaF3 is highly suited for the development of combination therapies targeting ALK-positive cancers. Experimental designs could include assessing the synergistic effects of combining ALK inhibitors with other therapeutic agents such as chemotherapy, immunotherapy, or targeted therapies against different pathways. Dose-response curves and apoptosis assays can be conducted to determine the optimal combination doses and to understand the mechanism of action when used in combination, thereby identifying potential therapeutic strategies for overcoming resistance mechanisms.
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The Human EML4-ALK-L1198F Stable Cell Line - BaF3 features the L1198F mutation in the ALK fusion gene, providing a targeted model for studying specific mutation effects on cell signaling and behavior. This specificity allows for detailed investigation of mutation-driven cellular responses, critical for understanding the impact of such mutations.
This cell line ensures reliable expression of the EML4-ALK fusion gene with the L1198F mutation, providing a stable genetic environment for consistent experimental outcomes. The Human EML4-ALK-L1198F Stable Cell Line - BaF3 is essential for maintaining fidelity in gene expression across various experimental setups.
Known for its robust growth, the BaF3 line ensures that the Human EML4-ALK-L1198F Stable Cell Line - BaF3 supports continuous and vigorous cell proliferation. This robust growth is advantageous for conducting prolonged studies without compromising cell health or experimental integrity.
The stable nature of the Human EML4-ALK-L1198F Stable Cell Line - BaF3 ensures enhanced precision in experimental data. This precision is crucial when assessing the specific contributions and impacts of the L1198F mutation in ALK, particularly in the context of targeted therapy development and molecular research.
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