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Intracellular calcium (Ca²⁺) regulates a wide range of cellular functions by activating various downstream cellular mediators. Elevated intracellular Ca²⁺ levels lead to the activation of calcineurin—a Ca²⁺- and calmodulin-dependent phosphatase. Calcineurin dephosphorylates and activates the Nuclear Factor of Activated T-cells (NFAT) family of transcription factors, which plays a pivotal role in immune function, inflammatory responses, angiogenesis, and cell proliferation and differentiation.

The NFAT family comprises five members: NFAT1, NFAT2, NFAT3, NFAT4, and NFAT5. With the exception of NFAT5, all other members of this family are regulated by changes in intracellular Ca²⁺ levels. NFAT proteins possess a conserved N-terminal domain containing multiple serine residues, which remain phosphorylated under resting conditions. The nuclear translocation of NFAT proteins is inhibited when this N-terminal regulatory domain is phosphorylated. Upon cellular stimulation, the dephosphorylation of these serine residues is mediated by the Ca²⁺-dependent phosphatase, calcineurin. This dephosphorylation event unmasks nuclear localization signals, thereby facilitating nuclear translocation and the transcriptional induction of NFAT-regulated genes. In response to calcineurin-mediated Ca²⁺ activation, NFAT proteins can either independently induce gene expression or act synergistically with other transcription factors to do so. NFAT proteins bind to and function synergistically with other transcription factors—such as Activator Protein 1 (AP-1)—to induce gene transcription. Through these interactions with other transcription factors, NFAT proteins are able to integrate signals originating from diverse pathways and execute a multitude of cellular functions. One of the critical functions of NFAT proteins within immune cells is the induction of inflammatory cytokine expression.

The NFAT-Luc THP-1 reporter gene drug target model accurately simulates the *in vivo* signal transduction processes involving NFAT; The principle is illustrated in the figure below.

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

Figure 2. Induction of NFAT activity by Cell Stimulation Cocktail in NFAT-Luc THP-1(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.