KC-6047

KU812-NFκB-Luc2-Cell-Line

61173

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Background of KU812-NFκB-Luc2-Cell-Line

NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. The protein encoded by IL-33 is a cytokine that binds to the IL1RL1/ST2 receptor. The encoded protein is involved in the maturation of Th2 cells and the activation of mast cells, basophils, eosinophils and natural killer cells. Several transcript variants encoding different isoforms have been found for IL-33.

Specifications

Catalog Number	KC-6047
Cell Line Name	KU812-NFκB-Luc2-Cell-Line
Clone Number	1B4
Host Cell Line	KU812
Description	Stable KU812 cell line expressing exogenous luciferase gene under the control of NFκB 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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS
Morphology	Myeloblast
Subculture	Split saturated culture 1:3-1:4 every 2-3 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 36 hours
Mycoplasma Status	Negative

Cell Line Generation

KU812-NFκB-Luc2 Cell Line was generated using a lentiviral vector expressing NFκB sequence.

Characterization

Figure 1. KU812-NFκB-Luc2 cell line was seed into the 96-well plate, and treated with IL-33 at a maximum concentration 10 ng/mL diluted 3.16-fold for 16 hours, then readout with Bright-Glo system.

Figure 2. KU812-NFκB-Luc2 cell line was seed into the 96-well plate, and treated with Astegolimab/TQC2938-hIgG2/IgG1 at a maximum concentration 20 µg/mL diluted 3.16-fold for 1 hour, and then IL-33 treatment in the concentrations of 10 ng/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 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 1:3-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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.

References

1.Beinke S, Robinson MJ, Hugunin M, Ley SC. Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IkappaB kinase-induced proteolysis of NF-kappaB1 p105. Mol Cell Biol. 2004 Nov;24(21):9658-67. doi: 10.1128/MCB.24.21.9658-9667.2004. PMID: 15485931; PMCID: PMC522219.
2.Hansen SK, Nerlov C, Zabel U, Verde P, Johnsen M, Baeuerle PA, Blasi F. A novel complex between the p65 subunit of NF-kappa B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene. EMBO J. 1992 Jan;11(1):205-13. doi: 10.1002/j.1460-2075.1992.tb05043.x. PMID: 1740106; PMCID: PMC556441.
3.Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, Zurawski G, Moshrefi M, Qin J, Li X, Gorman DM, Bazan JF, Kastelein RA. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity. 2005 Nov;23(5):479-90. doi: 10.1016/j.immuni.2005.09.015. PMID: 16286016.