Colony Stimulating factor 1 receptor (CSF1R), also named as macrophage colony-stimulating factor receptor (M-CSFR) and CD115, is a single pass type I membrane protein and functions as receptor for the cytokine of colony stimulating factor 1 (CSF1), the interaction of both molecules controls the production, differentiation and function of macrophages, and is also involved in the development and carcinogenesis of the mammary gland. Mutations in CSF1R are also associated with CML and type M4 AML. The identification of CSF1R as drug target has led to repaid development of the inhibitor of CSF1R in the treatment of cancer or inflammatory diseases, such as Pexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4 and cabiralizumab.
Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.
Cell Line Name:
Ba/F3 ETV6-CSF1R Cell Line
Host Cell Line:
Mouse Ba/F3 cell line
Stable Ba/F3 clone expressing exogenous human ETV6-CSF1R fusion gene
One vial of frozen cells (5X106 per vial)
Stable in culture over a minimum of 10 passages
Drug screening and biological assays
70% RPMI1640+20% FBS+10% DMSO
Mostly single, round (some polymorph) cells in suspension
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 20 hours
Cell Line Generation
Ba/F3 ETV6-CSF1R cell line was generated using a retrovirus vector expressing the human ETV6-CSF1R sequence.
Characterization using PCR sequencing and WB
1. Prewarm culture medium (RPMI1640 + 10%FBS) in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 105 cells/mL.
1. Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) 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×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 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
1. Irvine, Katharine M, Christopher J Burns, Andrew F Wilks, Stephen Su, David A Hume, and Matthew J Sweet. 2006. “A CSF-1 Receptor Kinase Inhibitor Targets Effector Functions and Inhibits Pro-Inflammatory Cytokine Production from Murine Macrophage Populations.” The FASEB Journal 20 (11): 1921–23.
2. Ries, Carola H, Michael A Cannarile, Sabine Hoves, Jörg Benz, Katharina Wartha, Valeria Runza, Flora Rey-Giraud, et al. 2014. “Targeting Tumor-Associated Macrophages with Anti-CSF-1R Antibody Reveals a Strategy for Cancer Therapy.” Cancer Cell 25 (6). Elsevier
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