KC-1271

Ba/F3-RBM6-CSF1R-Cell-Line

21833

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Background of Ba/F3-RBM6-CSF1R-Cell-Line

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.

Specifications

Catalog Number	KC-1271
Cell Line Name	Ba/F3-RBM6-CSF1R-Cell-Line
Description	Stable Ba/F3 clone expressing exogenous human RBM6 CSF1R fusion gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+1µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3 RBM6-CSF1R cell line was generated using a retrovirus vector expressing the human RBM6-CSF1R sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 1µg/mL Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Irvine, Katharine M, Christopher J Burns, Andrew F Wilks, Stephen Su, David A Hume, and Matthew J Sweet. 2006. ¡ùA CSF-1 Receptor Kinase Inhibitor Targets Effector Functions and Inhibits Pro-Inflammatory Cytokine Production from Murine Macrophage Populations.¡ì The FASEB Journal 20 (11): 1921Ã¿23.
Ries, Carola H, Michael A Cannarile, Sabine Hoves, J?rg Benz, Katharina Wartha, Valeria Runza, Flora Rey-Giraud, et al. 2014. ¡ùTargeting Tumor-Associated Macrophages with Anti-CSF-1R Antibody Reveals a Strategy for Cancer Therapy.¡ì Cancer Cell 25 (6). Elsevier