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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.

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

NFκB-Luc Jurkat reporter cells are Jurkat cells engineered to express a luciferase (Luc) reporter gene under the transcriptional control of NFκB. PMA is an analog of DAG (diacylglycerol) that can freely traverse the cell membrane and directly act upon PKC within the cellular activation signaling transduction pathway; the underlying mechanism is illustrated in the figure below.

Figure 1. Schematic diagram of the Jurkat E6.1 Human NFκB-Luc Cell model.

Figure 2. Induction of NF-κB activity by PMA with Ionomycin in NFκB-Luc Jurkat Cell(C1).

Cell Passage Procedures

1.This cell line grows in suspension.
2.Upon receipt, cells should be thawed immediately or stored in liquid nitrogen until use.
3.Before thawing, pre-warm the water bath and culture medium to 37 °C, and prepare a small amount of dry ice.
4.Remove the cryovial from storage and transport it to the cell culture laboratory on dry ice.
5.Rapidly thaw the cells in a 37 °C water bath. Once the cells are completely thawed, spray the cryovial with 70% ethanol for disinfection and transfer it to a biosafety cabinet.
6.Add 10 mL of pre-warmed culture medium into a 15 mL centrifuge tube. Transfer the contents of the cryovial into the tube and centrifuge at 1000 rpm for 5 minutes.
7.Carefully discard the supernatant. Resuspend the cell pellet in 5 mL of pre-warmed culture medium by gentle pipetting. Immediately perform cell counting and adjust the cell density to 3–6 × 10⁵ cells/mL based on the counting results, then transfer the cells into a culture flask.
8.Count the cells every 1–2 days. When the cell density exceeds 1 × 10⁶ cells/mL, passage the cells promptly or add fresh culture medium. Maintain the cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL.

Suspension Cell Cryopreservation Procedure:

1.Collect 8 × 10⁶ cells, centrifuge, and discard the supernatant.
2.Add 1 mL of cell freezing medium (90% FBS + 10% DMSO) and gently pipette to mix thoroughly. Transfer the suspension into a cryovial.
3.Immediately place the cryovial into a controlled-rate freezing container (Nalgene 5100-0001), fill with isopropanol up to the indicated level, and store at −80 °C.
4.After 24 hours, transfer the cryovial to liquid nitrogen for long-term storage.