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Toll-like receptors (TLRs) are highly conserved pattern recognition receptors (PRRs) capable of recognizing various types of pathogen-associated molecular patterns (PAMPs) derived from microorganisms. In humans, 10 members of the Toll-like receptor family (TLR1–10) have been identified, while 12 members (TLR1–9, TLR11, and TLR13) have been found in mice. TLR1, TLR2, TLR4, TLR5, and TLR6 are localized to the cell surface membrane; conversely, TLR3, TLR7, TLR8, TLR9, and TLR10 are localized to the membranes of intracellular endosomes (specifically, TLR3 recognizes dsRNA, and TLR9 recognizes dsDNA). The activation of downstream signaling pathways mediated by TLRs primarily relies on two classes of transcription factors: NF-κB and interferon regulatory factors (IRFs), which predominantly induce the production of pro-inflammatory cytokines and type I interferons (IFNs).

TLR7 is a critical pattern recognition receptor protein involved in the recognition of viral nucleic acids. The activation of TLR7 and TLR8 triggers the induction of Th1-type innate immune responses. The expression profile of TLR7 varies across different cell types and is subject to differential regulation. Compared to other circulating immune cells, the constitutive expression of TLR7 is most prominent in human and murine plasmacytoid dendritic cells (pDCs) and B cells. Sequence-specific single-stranded RNA (ssRNA) is considered the natural ligand for TLR7; upon activation, it induces the production of IFN-α, TNF-α, and IL-12. ssRNA sequences derived from viruses such as HIV-1, HBV, or influenza virus can induce pDCs to produce pro-inflammatory cytokines, thereby exerting an immune-enhancing effect. Furthermore, TLR7 is considered a potential therapeutic target for cancer treatment; for instance, the TLR7/8 agonist R848 has been shown to induce anti-tumor responses and ameliorate cachexia. The principle by which TLR7 agonists exert their therapeutic effects against tumors involves stimulating plasmacytoid dendritic cells (pDCs) to secrete IFN-α, which subsequently acts upon other immune cells (such as NK cells and macrophages) to enhance immune responses. Simultaneously, these agonists activate pDCs—thereby augmenting their antigen-presenting capabilities—and promote the proliferation of CD4+ T cells; this, in turn, leads to the further activation of CD8+ T cells, facilitating the elimination of tumor cells and ultimately exerting an anti-tumor effect.

The TLR7 NFκB & ISRE Dual Reporter Cell model serves as an excellent *in vitro* surrogate for the signal transduction processes of TLR7 observed *in vivo*; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human TLR7 NFκB&ISRE Dual Reporter Cell Model

Figure 2.NFκB Reporter Activity induced by R848 in TLR7 NFκB&ISRE Dual Reporter Cell(C3).

Figure 3. ISRE Reporter Activity induced by R848 in TLR7 NFκB&ISRE Dual Reporter Cell(C3).

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 TLR7 NFκB&ISRE Dual 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.