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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 microbial pathogens. 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); these factors predominantly induce the production of pro-inflammatory cytokines and Type I interferons (IFNs).

TLR3 plays a pivotal role in antiviral defense by inducing robust immune responses against the dsRNA generated during viral infections. Several synthetic dsRNA molecules are known to activate TLR3. Among these, poly(I:C) stands out as one of the most potent immune inducers for TLR3 activation, capable of triggering the production of IFN-α/β and the activation of NK cells. Upon binding to dsRNA, the adaptor protein TRIF is recruited to the dimerized TLR3 receptor. This interaction leads to the activation of Interferon Regulatory Factor-3 (IRF-3), NF-κB, and AP-1, thereby driving the induction of IFNs—particularly IFN-β—as well as the production of various cytokines and chemokines, and the maturation of dendritic cells.

The TLR3 NFκB-Luc HEK293 reporter gene drug target model accurately recapitulates the in vivo signal transduction processes mediated by TLR3;  The principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the TLR3/NFκB-Luc HEK293 Cell Model

Figure 2. Dose Response of Poly(I:C) in TLR3 NFκB-Luc HEK293(C7).

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 TLR3/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.