Cat.No. : CSC-RO0120 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-RO0120 |
| Description | Ba/F3-EML4-ALK cell line is a stably transfected cell line which expresses human EML4-ALK fusion protein. |
| 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 |
| 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 oncogenic echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) (EML4-ALK) fusion protein found in non-small cell lung cancer (NSCLC) dimerizes through the coiled-coil domain (cc) within EML4 and is thus constitutively phosphorylated and activated. Here, researchers investigated whether disruption of ALK fusion protein oligomerization by competing cc-mimetic compounds could be a therapeutic strategy for EML4-ALK NSCLC. Cells induced to express monomeric ALK in vitro were unable to survive. When transplanted into mice, induction of monomers abolished tumor formation. Using a fluorescent protein system to quantify protein-protein interactions of EML4-ALK and EML4cc, researchers demonstrated that co-expression of EML4cc inhibited EML4-ALK assembly and reduced tumor growth rates both in vitro and in vivo. In EML4-ALK cancer cell lines, administration of a synthetic EML4cc peptide reduced phosphorylation of ALK, thereby reducing tumor cell growth. These findings support the use of EML4cc peptide to monomerize ALK fusion protein, thereby competitively inhibiting dimerization, as a promising therapeutic strategy for EML4-ALK NSCLC.
To investigate the inhibitory role of the endogenous EML4 coiled-coil (cc) domain in Ba/F3 expressing EML4-ALK, the researchers used FluoppiTM technology to create an in vitro fluorescent model of protein-protein interaction. Ba/F3 EA/EA cells expressing hAG-tagged EML4-ALK variant 1 (EA) and Ash-tagged EA constructs were co-transfected in Ba/F3 cells, while Ba/F3 EA/cc cells expressing hAG-tagged EA and Ash-tagged cc constructs were co-transfected in Ba/F3 cells. FluoppiTM assays revealed physical interactions between EML4-ALK and EML4-ALK or EML4-ALK and EML4cc (Figure 1A). The study showed that homotetrameric Azami-GFP-EA protein was the main population, while Azami-GFP hyper, reflecting the interaction between Azami-GFP-EA protein and Ash-EML4cc protein, was only a small part of the interaction 48 hours after gene introduction (Figure 1B). In the MTS cell growth assay, the growth of Ba/F3 EA/cc cells was significantly lower than that of Ba/F3 EA/EA, indicating that the oligomerization of EA expressed by endogenous cc can inhibit tumor growth (Figure 1C). In the xenograft model, both Ba/F3 EA/EA and Ba/F3 EA/cc were inoculated to form tumors. The tumor growth delay in the Ba/F3 EA/cc group was more obvious than that in the EA/EA group (Figure 1D).
Figure 1. Growth inhibition of endogenous EML4-coiled-coil domain in Ba/F3 cells expressing EML4-ALK. (Hirai N, et al., 2020)
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The Human EML4-ALK Stable Cell Line-Ba/F3 has significantly improved the reliability of our kinase inhibitor testing. The consistency and stability of these cells provide us with reliable and reproducible data, which is critical for our drug development programs.
These cells have proven to be extremely versatile in a variety of applications in our laboratory. From drug screening to mechanistic studies, the Human EML4-ALK Stable Cell Line-Ba/F3 has been a reliable and indispensable tool in our experiments.
The detailed protocols and user-friendly handling instructions provided with the Human EML4-ALK Stable Cell Line-Ba/F3 have been incredibly helpful. Even with minimal previous experience, our team was able to get the cells up and running quickly, ensuring smooth integration into our existing projects.
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