Cat.No. : CSC-RO0428 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-RO0428 |
| Description | This cell line is engineered to stably overexpress exogenous human TPR-MET fusion protein. |
| Target Gene | TPR-MET |
| 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 |
MET-targeted therapies are effective in MET-amplified and METex14 NSCLC, but face drug resistance challenges. Researchers assessed MET tyrosine kinase inhibitors (TKIs) in overcoming drug resistance in NSCLC. Using TPR-MET transformed Ba/F3 cell mutagenesis assays, they evaluated secondary MET mutations after TKI treatments. While individual TKIs showed distinct mutation profiles, combining type I/II TKIs (capmatinib and merestinib) resulted in no resistant clones in vitro. In vivo, this combination significantly reduced tumor growth compared to single TKIs. Simultaneous use of type I and type II MET TKIs may delay or diminish drug resistance mutations, offering a promising clinical approach.
Figure 1. Using the Human TPR-MET Stable Cell Line, distinct secondary MET mutations upon MET TKI treatment were studied. Mutagenized cells were treated until resistant clones emerged, facilitating clone counting. Sequencing revealed secondary MET TKD mutations, aiding in understanding drug resistance mechanisms. (Bahcall M, et al., 2022)
A: BaF3 cells were chosen for establishing the stable cell line expressing human TPR-MET fusion protein due to their lack of endogenous MET expression and their responsiveness to MET activation, providing a clean background for studying TPR-MET signaling. BaF3 cells also offer a suitable environment for investigating the oncogenic potential and drug sensitivity of TPR-MET.
A: The stability and expression level of human TPR-MET in the BaF3 stable cell line was confirmed and sustained through stable transfection techniques, followed by clonal selection to isolate cells with stable integration of TPR-MET. Expression of TPR-MET was validated using qPCR and western blot analysis. Continuous culture in the presence of appropriate selection agents ensured the maintenance of stable expression levels.
A: Functional characterization of human TPR-MET in the BaF3 stable cell line focused on its kinase activity and downstream signaling effects. This involved assessing the phosphorylation of key signaling molecules such as MAPK and AKT using western blotting. Additionally, cell proliferation and migration assays were conducted to evaluate the oncogenic potential and downstream effects of TPR-MET activation.
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Rock-solid stability. The Human TPR-MET Stable Cell Line in Ba/F3 cells keeps TPR-MET fusion protein expression consistent, ensuring reliable results in cancer research. With stable TPR-MET expression, I'm digging into oncogenic signaling pathways confidently, deepening our understanding of cancer biology.
Dependable to the core. This cell line exceeds expectations, giving me a sturdy platform for studying TPR-MET-targeted therapies and MET-driven cancers. It's supercharged my research, offering valuable insights into TPR-MET-mediated oncogenesis and potential cancer treatments.
Its stable expression makes experiments easier, speeding up data collection and analysis for breakthroughs in cancer pathogenesis.
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