Cat.No. : CSC-RO1014 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-RO1014 |
| Description | Ba/F3-EML4-ALK-G1202R-L1196M cell line is engineered to stably overexpress human EML4-ALK fusion protein with G1202R-L1196M point 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 |
Alectinib has shown higher efficacy in first-line treatment of ALK-rearranged non-small cell lung cancer. However, most patients relapse due to acquired resistance, such as secondary mutations of ALK, including I1171N and G1202R. Although ceritinib or lorlatinib have been shown to be effective against these resistance mutants, relapsed patients often develop further resistance due to ALK compound mutations after the use of ceritinib or lorlatinib. Here, researchers identified 14 lorlatinib-resistant ALK compound mutants, including several mutants recently found in lorlatinib-resistant patients. Some of these compound mutants were found to be sensitive to early ALK-TKIs and several BCR-ABL inhibitors. Using computational simulations, researchers successfully demonstrated a clear linear correlation between the binding free energy and in vitro experimental IC50 values of several ALK-TKIs with single or compound mutant EML4-ALK-expressing Ba/F3 cells, and reproduced the trend of reduced binding affinity caused by double mutations found in the study. Computational simulations revealed that the ALK-L1256F single mutant conferred resistance to lorlatinib but increased sensitivity to alectinib.
The results showed that AG-957, developed as a BCR-ABL inhibitor, preferentially inhibited the viability of G1202R+L1196M cells, rather than ALK-WT or ALK-G1202R mutant cells (Figure 1a and b). Therefore, to study the sensitivity to AG-957 in detail, they selected BaF3 cells #50 expressing EML4-ALK-G1202R+L1196M, whose sensitivity to the HSP90 inhibitor 17-AAG was similar to that of Ba/F3 EML4-ALK wild-type, Ba/F3 G1202R, and G1202R+L1196M polyclonal cells. Next, the detailed sensitivity of EML4-ALK-G1202R+L1196M #50 cells to AG-957 and adaphostin, an AG-957 analog, was tested (Figure 1c). Compared with ALK-G1202R or ALK-WT cells, EML4-ALK-G1202R+L1196M cells #50 showed lower IC50 values (Figure 1d-g). Since AG-957 and adaphostin treatment significantly reduced ALK autophosphorylation and phosphorylation of downstream signals in a dose-dependent manner, it was suggested that AG-957 and adaphostin directly inhibited the ALK-G1202R+L1196M mutation (Figure 1h).
Figure 1. Identification of inhibitors able to inhibit the lorlatinib-resistant ALK-G1202R + L1196M double mutant. (Okada K, et al. 2019)
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The Human EML4-ALK-G1202R-L1196M Stable Cell Line-Ba/F3 has significantly enhanced our drug screening assays. The high stability and reproducibility of this cell line have provided us with reliable data, accelerating our research and development process.
As researchers focused on ALK-related cancer therapies, this cell line has been invaluable. It closely mimics the in vivo environment, enabling us to test ALK inhibitors more effectively. This product has definitely advanced our experimental models and paved the way for new therapeutic discoveries.
This product was easy to integrate into our laboratory workflow thanks to the detailed support documentation provided. The clear instructions helped us grow and maintain the cell line with minimal hassle, allowing us to focus more on our experimental goals.
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