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Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a family of transcription factor protein complexes that controls DNA transcription, cytokine production, and cell survival. NF-κB is present in almost all animal cell types and participates in cellular responses to stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens.

NF-κB activation involves two major signaling pathways: the canonical pathway and the non-canonical (or alternative) pathway. Although their signaling mechanisms differ, both are crucial for regulating immune and inflammatory responses. The primary mechanism of canonical NF-κB activation involves the inducible degradation of IκBα, triggered by site-specific phosphorylation mediated by the multi-subunit IκB kinase (IKK) complex. In contrast to the canonical pathway, the non-canonical NF-κB pathway responds selectively to a specific subset of stimuli—specifically, ligands for certain members of the TNFR superfamily, such as LTβR, BAFFR, CD40, and RANK.

NF-κB activation is initiated when molecules such as TNFα bind to TNF receptors (of which there are various types). Once a TNF receptor is activated, a complex signal transduction cascade ensues; the IκB kinase (IKK) is ultimately triggered, leading to the phosphorylation of IκB, which in turn results in the ubiquitination and degradation of IκB. Upon the degradation of IκB, the remaining NF-κB dimers (e.g., p65/p50 or p52/p50 subunits) translocate into the cell nucleus, where they bind to consensus DNA sequences within target genes.

NFκB-Luc THP-1 reporter cells are THP-1 cells engineered to express a luciferase (Luc) reporter gene under the transcriptional control of NF-κB. The mechanism by which TNFα binding to its receptor activates NF-κB is illustrated in the figure below.

Figure 1. Schematic Diagram of the NFκB-Luc THP-1 Principle

Figure 2. Dose Response of Recombinant Human TNFα in NFκB-Luc THP-1(C1).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed THP-1 Human NFκB-Luc Reporter Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.