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Signal Transducers and Activators of Transcription (STATs) have been identified as a major class of DNA-binding proteins capable of activating gene transcription in response to a wide variety of cytokines. The seven distinct members of the STAT family—STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6—regulate numerous physiological and pathological processes, ranging from pathogen responses to cytokine secretion. STAT4 is activated by a diverse array of cytokines, including IL-12, Type I Interferons (IFN-I), IL-23, IL-2, IL-27, IL-35, and others.

IFN Signaling Pathway: IFNα and IFNγ bind to their respective receptors—IFNαR and IFNγR—thereby activating JAK1/TYK2 (in the case of IFNαR) or JAK2 (in the case of IFNγR). This activation leads to the phosphorylation of STAT proteins, which subsequently translocate into the cell nucleus to bind to conserved DNA elements. Type II Interferons typically act as specific activators of STAT1, whereas Type I Interferons activate both STAT1 and STAT2; both types of interferons can subsequently lead to the activation of STAT3 and STAT4.

STAT4-Luc HEK293 Reporter Cells are a line of HEK293 cells engineered to express a Luciferase (Luc) reporter gene under the transcriptional control of STAT4. The mechanism by which IFNγ binds to its receptor and activates STAT4 is illustrated in the figure below.

Figure 1. Schematic Diagram of the STAT4-Luc HEK293 Principle

Figure 2. Dose Response of Recombinant Human IFNγ in STAT4-Luc HEK293(C9).

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 HEK293 Human STAT4-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.