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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, both type I and type II receptors are activated, leading to the dimerization and rearrangement of receptor subunits, thereby activating associated JAKs through autophosphorylation and further activating STAT proteins. After phosphorylation, the activated STATs form homodimers and then translocate to the cell nucleus, where they initiate the transcription of IFN-stimulated genes.

      Type I interferon also induces the formation of the ISGF3 complex, which consists of STAT1, STAT2, and IRF9. The ISGF3 complex binds to the ISRE (interferon-stimulated response element), further inducing the transcription of interferon-stimulated genes containing ISREs within their promoters.

      Binding of IFNγ to the IFNγ receptor leads to tyrosine phosphorylation of 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 THP-1 reporter cells are THP-1 cells in which the GAS gene regulates the expression of the Luc luciferase reporter gene. The mechanism by which IFN binds to its receptor and activates GAS is shown in the figure below.

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

Figure 2. Dose Response of Recombinant Human IFN in GAS-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.