Cat.No. : CSC-RO0381 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-RO0381 |
| Description | This cell line is engineered to stably overexpress exogenous human FLT3 with ITD duplicate and D835V mutations. |
| Target Gene | FLT3-ITD |
| 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 |
Researchers demonstrate the synergistic effect of JQ1, a BET protein antagonist, and FLT3 tyrosine kinase inhibitors (TKIs) like ponatinib or AC220 in inducing apoptosis of human AML blast progenitor cells (BPCs) expressing FLT3-ITD. Co-treatment led to significant downregulation of c-MYC, BCL2, and CDK4/6 while upregulating p21, BIM, and cleaved PARP levels. Additionally, it decreased phosphorylation levels of STAT5, AKT, and ERK1/2. Notably, this combination therapy showed minimal activity against normal bone marrow progenitor cells. Knockdown of BRD4 sensitized AML cells to FLT3-TKIs. JQ1 also induced apoptosis in FLT3-ITD-expressing cells, including those with FLT3-TKI-resistant mutations. These findings support further investigation of combined BA and FLT3-TKI or BA and HDI therapy against AML cells, particularly those resistant to FLT3-TKIs.
Figure 1. The efficacy of JQ1 against BaF3 cells stably expressing FLT3-ITD or FLT3-ITD along with FLT3-F691L or FLT3-D835V mutations was investigated by researchers. Apoptosis was induced in all cell types by JQ1, with varying IC50 values. This effect was associated with c-MYC attenuation and induction of HEXIM1 and BIM isoforms. The effectiveness of JQ1 against cells expressing clinically relevant FLT3 mutations conferring resistance to FLT3-TKI therapy was maintained. (Fiskus W, et al., 2014)
A: BaF3 cells were selected for establishing the stable cell line expressing human FLT3-ITD-D835V based on several considerations. BaF3 cells are an interleukin-3 (IL-3)-dependent murine pro-B cell line commonly used to study the oncogenic properties of mutated FLT3 receptor tyrosine kinase, such as FLT3-ITD-D835V. BaF3 cells lack endogenous FLT3 expression, providing a clean background for studying the effects of exogenous expression of FLT3 mutants. Additionally, BaF3 cells are highly proliferative and amenable to genetic manipulation, making them suitable for establishing stable cell lines expressing FLT3 mutants for functional studies.
A: Several approaches were employed to verify and maintain the stability and expression level of human FLT3-ITD-D835V in the BaF3 stable cell line. Stable transfection techniques were used to introduce the FLT3-ITD-D835V gene into BaF3 cells, followed by selection with appropriate antibiotics to isolate cells with stable integration of the gene. The clonal selection was performed to obtain cell lines with uniform and stable expression levels of FLT3-ITD-D835V. Expression of FLT3-ITD-D835V was confirmed at both mRNA and protein levels using qPCR and western blot analysis, respectively. Additionally, functional assays such as cell proliferation assays and phosphorylation assays were conducted to validate the oncogenic activity and kinase activity of FLT3-ITD-D835V in the BaF3 stable cell line.
A: The functional characterization of human FLT3-ITD-D835V in the BaF3 stable cell line focused on assessing its kinase activity and downstream signaling effects. FLT3-ITD-D835V kinase activity was evaluated using in vitro kinase assays, where the phosphorylation of substrate proteins by FLT3-ITD-D835V was measured in the presence of ATP and inhibitors or activators of FLT3 kinase activity. Downstream signaling effects of FLT3-ITD-D835V were investigated by analyzing the phosphorylation status of key signaling molecules such as STAT5, AKT, and ERK using western blotting or immunofluorescence microscopy. Additionally, functional assays such as cell proliferation assays and apoptosis assays were conducted to assess the oncogenic potential of FLT3-ITD-D835V and its effects on cell survival and proliferation in the BaF3 stable cell line.
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This cell line is a game-changer, helping me explore FLT3-ITD-D835V-targeted therapies and personalized treatments for FLT3-mutant leukemias. It's supercharged my research, revealing insights into leukemia and potential treatment strategies.
It's all about optimizing workflows! Its stable expression streamlines experiments, making data collection and analysis more efficient, and pushing forward our understanding of leukemia and drug responses.
Consistent as can be. The Human FLT3-ITD-D835V Stable Cell Line in Ba/F3 cells keeps FLT3 mutations consistent, giving me trustworthy results in leukemia research. With stable FLT3-ITD-D835V expression, I'm delving into drug resistance mechanisms and new treatment strategies with confidence, unlocking the mysteries of leukemia biology.
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