KC-3473

RAW264.7-NFκB-Luc2-Cell-Line

Stable RAW264.7 cell line expressing exogenous luciferase under the control of NF-κB signaling pathway

34414

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Background of RAW264.7-NFκB-Luc2-Cell-Line

NF-κB pathway plays an important role in controlling many biological processes including transcription of DNA, cytokine production, cell survival, immune responses to infection, developmental processes, synaptic plasticity, memory and cancer development. In response to the various stimuli, such as stress, cytokines, free radicals, heavy metals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens, NFκB pathway is activated and NFκB protein complex translocate from cytoplasm to nucleus, regulates a wide spectrum of gene expression after binds to its response element on the promoter region of downstream gene.

Specifications

Catalog Number	KC-3473
Cell Line Name	RAW264.7-NFκB-Luc2-Cell-Line
Description	Stable RAW264.7 cell line expressing exogenous luciferase under the control of NF-κB signaling pathway
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% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS+400µg/mL HygromycinB
Selection Marker	HygromycinB
Morphology	Monocyte/Macrophage
Subculture	Split saturated culture 1:4-1:8 every 2 days; seed out at about 1-2 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 12 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

RAW264.7-NFκB-Luc2 Cell Line was generated using a lentiviral vector expressing luciferase under the control of NF-κB signaling pathway

Characterization

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 300µg/mL Hygromycin B) 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 1:4-1:8 every 2 days; seed out at about 1-2 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Gilmore TD (October 2006). "Introduction to NF-kappaB: players, pathways, perspectives". Oncogene. 25 (51): 6680ÿ4.
Brasier AR (2006). "The NF-kappaB regulatory network". Cardiovascular Toxicology. 6 (2): 111ÿ30.