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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).

TLR2 is unique among TLRs in its ability to form functional heterodimers with more than two other types of TLRs; it forms dimers with TLR1 and TLR6, and in certain contexts, with TLR4. TLR2 also interacts with various non-TLR molecules, thereby enabling the recognition of a wide array of PAMPs derived from all classes of microorganisms—including viruses, fungi, bacteria, and parasites. TLR2 is capable of sensing highly conserved lipoproteins expressed on the outer membranes of Gram-positive bacteria, as well as certain membrane antigens from Gram-negative bacteria—such as lipopolysaccharide (LPS)—in conjunction with its co-receptor, CD14.

TLR2 relies primarily on the adaptor proteins MyD88 and TIRAP to mediate signal transduction. The MyD88-dependent pathway involves interactions between death domains that mediate intracellular signaling in a stepwise fashion. Specifically, the recruitment of MyD88 is indirect, being mediated by TIRAP. Subsequently, the activated MyD88 sequentially triggers the phosphorylation of IRAK4, IRAK1, and IRAK2. The IRAK complex binds to TRAF6, thereby activating a complex comprising TAK1, TAB2, and TAB3. TAK1 subsequently phosphorylates IKKα and IKKβ; the IKK complex then phosphorylates IκB, marking it for degradation. This process ultimately leads to the production of pro-inflammatory cytokines via NF-κB and AP-1 pathways, as well as the activation of MAPKs, which regulate cellular proliferation and survival.

The TLR2 NFκB-Luc HEK293 reporter gene drug target model accurately recapitulates the in vivo signal transduction processes of TLR2. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the TLR2/NFκB-Luc Cell Model

Figure 2.Dose Response of Pam3CSK4 in TLR2 NFκB-Luc HEK293(C5).

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 TLR2/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.