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

TLR4 is capable of activating both innate and adaptive immune cells. Activation of TLR4 by LPS or DAMPs triggers the production of pro-inflammatory cytokines via either MyD88-dependent or MyD88-independent signaling pathways. TLR4 plays multifaceted roles in a wide range of pathological conditions, including cardiovascular diseases, allergic disorders, obesity-related metabolic diseases, neurodegeneration, apoptosis, autoimmune diseases, infectious diseases, and inflammatory bowel disease.

The TLR4-NFκB-Luc HEK293 reporter gene model serves as an excellent *in vitro* surrogate for the *in vivo* signal transduction processes mediated by TLR4; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human TLR4/NFκB-Luc Cell Model

Figure 2.Dose Response of LPS-EB in TLR4 NFκB-Luc HEK293(C4).

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 TLR4/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.