Cat.No. : CSC-RO0417 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-RO0417 |
| Description | This cell line is engineered to stably overexpress exogenous human CSF1R protein. |
| Target Gene | CSF1R |
| 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 |
Colony-stimulating factor-1 receptor (CSF1R) is a receptor tyrosine kinase that controls the differentiation and maintenance of most tissue-resident macrophages. Inhibition of CSF1R has been considered a potential therapeutic approach for a variety of human diseases. Here, researchers report the synthesis, development, and structure-activity relationship of a series of highly selective pyrrolo[2,3-d]pyrimidine compounds. These compounds exhibit sub-nanomolar inhibitory activity against this receptor and excellent selectivity for other kinases in the platelet-derived growth factor receptor (PDGFR) family. The crystal structures of the protein and compound 23 indicate that the protein's binding conformation is similar to DFG-out. Cellular activity analysis, pharmacokinetic analysis, and in vivo stability studies were performed on the most promising compounds in this series, suggesting their potential therapeutic value for various diseases. Furthermore, these compounds primarily inhibit the autoinhibitory form of the receptor, contrasting with the behavior of pexidartinib, which may explain the excellent selectivity of these structures.
Here, researchers performed two cell-based assays on some compounds: one using genetically engineered Ba/F3 cells dependent on CSF1R activation, and the other using mouse bone marrow-derived macrophages. Disappointingly, the potency of these compounds showed a low correlation with the potency observed in the CSF1R kinase activity assay. However, in the mouse bone marrow-derived macrophage activity assay, some compounds showed activity comparable to pexidartinib. The Ba/F3 cell activity assay showed superior activity of pexidartinib, while some compounds that performed well in the kinase activity assay showed no activity. Among these new inhibitors, the metabolically unstable 3-pyridyl compound 39 showed the best inhibitory effect on CSF1R-overexpressing Ba/F3 cells. Unfortunately, when interleukin-3 was administered concurrently as a toxicity control, both phenol 36 and 3-pyridyl compound 39 severely affected CSF1R-overexpressing Ba/F3 cell viability. The efflux ratio also clearly indicated that the carboxylic acid group was highly unfavorable, which may also be one of the reasons for their inactivity in the Ba/F3 assay.
Figure 1. Cellular potency of selected compounds toward CSF1R-overexpressing Ba/F3
cells, bone marrow-derived
macrophages from mice, osteoclast differentiation, and the corresponding Caco2-Efflux ratio. (Aarhus T I, et
al., 2023)
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