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 Interferon (IFN) is a multifunctional soluble glycoprotein produced by monocytes and lymphocytes, and serves as a key effector molecule in both innate and adaptive immunity. IFN cytokines are classified into three types—Type I, Type II, and Type III—based on the specific interferon receptors they bind to. Type I interferons comprise a diverse group, including IFN-α, IFN-β, IFN-δ, IFN-κ, IFN-ω, IFN-τ, IFN-ε, and IFN-ξ; Type II interferon is IFN-γ; Type III interferon is IFN-λ, which includes IFN-λ1, IFN-λ2, and IFN-λ3. The primary functions of Type I interferons are antiviral and antitumor activity, while Type II interferons primarily induce the expression of major histocompatibility complex (MHC) antigens and exert immunomodulatory effects; however, their antiviral activity is weaker than that of Type I interferons. Type III interferons are a new class of interferons discovered in 2003, possessing broad-spectrum antiviral and immunomodulatory activities.

      Upon ligand binding, the type II receptor is activated, leading to receptor subunit dimerization and rearrangement, thereby activating the associated JAK through autophosphorylation and further activating STAT proteins. After phosphorylation, the activated STAT forms homodimers, which then translocate to the cell nucleus, where they initiate the transcription of IFN-stimulated genes. The binding of IFNγ to the IFNγ receptor leads to the tyrosine phosphorylation of downstream STAT1 at the Tyr701 site. The phosphorylated STAT1 homodimer translocates to the cell nucleus and binds to the GAS (IFNγ activation site) element, thereby inducing the expression of IFNγ-regulated genes.

GAS-Luc HEK293 reporter cells are HEK293 cells in which the GAS gene regulates the expression of the Luc luciferase reporter gene. The mechanism by which IFNγ binds to its receptor and activates GAS is shown in the figure below.

Figure 1. Schematic diagram of the GAS-Luc HEK293 cell model

Figure 2. Dose Response of Recombinant Human IFNγ in GAS-Luc HEK293(C6).

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 GAS-Luc 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.