Cat.No. : CSC-RO0401 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-RO0401 |
| Description | This cell line is engineered to stably overexpress exogenous human KRAS with G12D mutation. |
| Target Gene | KRAS |
| 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 have identified that mutations in RAS pathway genes are common in acute lymphoblastic leukemia (ALL), yet the specific impacts of these mutations, particularly the KRAS-G12D mutation, on ALL cell proliferation remain inadequately characterized. In their study, they utilized Reh ALL cells with the KRAS-G12D mutation, observing that these cells exhibited increased proliferation rates in vitro but showed significantly impaired growth in mouse models. Further investigation revealed that KRAS-G12D altered the metabolism of methionine and arginine, enhancing their catabolism to support the synthesis of polyamines and proline. This metabolic shift was critical, as inhibiting polyamine biosynthesis selectively induced cell death in KRAS-G12D-positive B-ALL cells. Additionally, blocking AKT/mTOR signaling restored amino acid metabolism and boosted the in vivo growth of these cells, highlighting the distinct effects of hyperactivated signaling in hematological malignancies versus solid tumors.
Figure 1. The researchers employed growth curves of KRAS-G12D-expressing Reh and BaF3 cells under nutrient-limited conditions to assess their proliferation and metabolic adaptations. (Xu Y, et al., 2022)
Our Human KRAS-G12D Stable Cell Line - BaF3 serves as a powerful tool for researchers investigating the mechanisms behind RAS mutations and their therapeutic implications in B-ALL. Creative Biogene's stable cell line can be utilized to explore the effects of KRAS-G12D on cellular metabolism and drug sensitivity, facilitating the development of targeted therapies.
A: The Human KRAS-G12D Stable Cell Line - BaF3 is instrumental in cancer research as it harbors the KRAS-G12D mutation, one of the most common KRAS mutations in human cancers. This cell line enables researchers to study the molecular and cellular mechanisms driven by this mutation in various cancers, including pancreatic, colorectal, and lung cancers. It is particularly useful for evaluating the efficacy of targeted therapies that inhibit KRAS-G12D-mediated signaling pathways, providing valuable insights into the development of precision medicine strategies.
A: When assessing the response of the Human KRAS-G12D Stable Cell Line - BaF3 to anti-cancer compounds, it's critical to include appropriate controls, such as untreated cells and cells treated with known inhibitors of KRAS signaling, to validate the experimental setup. Dose-response curves should be generated to determine the IC50 values of the compounds. Additionally, it's important to monitor the activation status of downstream signaling pathways, such as ERK and AKT, using Western blot or ELISA, to confirm the mechanism of action of the compounds.
A: To ensure the preservation of the KRAS-G12D mutation within the Human KRAS-G12D Stable Cell Line - BaF3, researchers should perform regular genotyping assays, such as PCR followed by sequencing, to verify the presence of the mutation. It's also advisable to maintain the cells under selective pressure by including appropriate antibiotics in the culture medium and to avoid over-confluency, which can lead to selective pressures that may cause loss of the mutation or emergence of dominant clones without the mutation.
A: The Human KRAS-G12D Stable Cell Line - BaF3 can be co-cultured with other cell types, such as fibroblasts, immune cells, or endothelial cells, to study the interaction between KRAS-driven cancer cells and the tumor microenvironment. These co-culture systems can help researchers understand how the microenvironment influences cancer progression, drug resistance, and metastasis in KRAS-mutant cancers. Analysis of cytokine profiles, cell migration, and invasion assays, as well as imaging techniques, can provide insights into these interactions.
A: To investigate the effect of novel KRAS inhibitors, it's recommended to use a combination of methodologies that assess both cell signaling alterations and apoptosis. Western blotting or ELISA can be employed to measure changes in the phosphorylation levels of key signaling molecules downstream of KRAS, such as MEK, ERK, and PI3K/AKT. For apoptosis, assays such as Annexin V/PI staining followed by flow cytometry, caspase activity assays, or TUNEL assay can quantify apoptotic cell death. These methodologies provide a comprehensive understanding of how KRAS inhibitors impact KRAS-G12D mutant cells.
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The Human KRAS-G12D Stable Cell Line - BaF3 specifically models the KRAS G12D mutation, prevalent in various cancers. This precision allows us to study the direct effects of this mutation on cellular processes, providing insights critical for understanding oncogenic signaling pathways.
This cell line ensures consistent expression of the KRAS G12D mutation, maintaining stable protein levels for reliable experimentation. The Human KRAS-G12D Stable Cell Line - BaF3 is essential for experiments requiring uniform mutant expression to achieve reproducible results.
The BaF3 background offers robust cell viability, which is crucial for the Human KRAS-G12D Stable Cell Line - BaF3. This feature supports extended experimental runs and complex study designs without compromising cell health, ensuring continuous data collection.
With the KRAS mutation already integrated, the Human KRAS-G12D Stable Cell Line - BaF3 provides enhanced experimental control, allowing us to focus on variables other than gene expression. This control is vital for dissecting the nuanced effects of the KRAS G12D mutation under various experimental conditions.
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