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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, while 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).

TLR8 is currently a subject of extensive research and a frequent target in drug development applications. Primarily expressed in monocytes, macrophages, and myeloid dendritic cells, TLR8 serves as a critical therapeutic target for enhancing host immune responses, facilitating cancer immunotherapy, and treating infectious diseases. The development of TLR8 agonists represents a major focus in current pharmaceutical R&D, with several such agonists having already advanced to the clinical development stage. Under physiological conditions, TLR8 is primarily activated by pathogen-derived RNA; however, numerous synthetic compounds have now been demonstrated to function as potent TLR8 agonists. In the context of antiviral defense, TLR8 participates in multiple stages of the antiviral response. TLR8 agonists activate the innate immune system, initiate intracellular signaling cascades, and induce the release of IFNs and other cytokines; by serving as a bridge between innate and adaptive immunity, these agonists hold promise as an immunotherapeutic strategy for treating viral infections. Furthermore, TLR8 agonists demonstrate significant therapeutic potential in oncology; their objective is to induce anti-tumor immune responses—exerting their effects by activating dendritic cells and natural killer (NK) cells, as well as by modulating T-cell activation. Additionally, these agonists can enhance immune responses to therapeutic antibodies—a benefit particularly relevant for individuals exhibiting diminished Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) activity.   

The TLR8 NFκB-Luc HEK293 reporter gene drug target model effectively mimics the in vivo signal transduction process of TLR8;  The principle is illustrated in the figure below.

Figure 1. Schematic diagram of the TLR8 NFκB-Luc HEK293 cell model

Figure 2. Dose response of Ligands in TLR8 NFκB-Luc HEK293(C1).

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 TLR8/NFκB-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.