Committed to providing comprehensive support for global drug development

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 regulatory 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 exposes 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 CHO-K1 reporter gene drug target model accurately simulates the in vivo signal transduction processes of NFAT; the underlying mechanism is illustrated in the figure below.

Figure 1. Schematic Diagram of the NFAT-Luc CHO-K1 Principle

Figure 2. Induction of NFAT Reporter Activity by PMA/Ionomycin Mixture in NFAT-Luc CHO-K1(C21).

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 CHO-K1 Human NFAT-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.