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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 have been identified (TLR1–10), while 12 members have been found in mice (TLR1–9, TLR11, and TLR13). 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).  

Upon recognizing PAMPs, the dimerization of the extracellular domain of TLR9 facilitates intracellular signal transduction, thereby recruiting the requisite adaptor proteins. TLR9 transmits downstream signals via a MyD88-dependent pathway. Adaptor proteins—such as MyD88 and IRAK4—activate the NF-κB and IRF signaling pathways, ultimately leading to the cellular production of pro-inflammatory factors and interferons.

The TLR9 NF-κB-Luc HEK293 reporter gene model accurately recapitulates the *in vivo* signal transduction process of TLR9; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the TLR9/NFκB-Luc HEK293 Cell Model

Figure 2.Dose Response of ODN 2006 in TLR9 NFκB-Luc HEK293(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 TLR9/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.