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OX40 (also known as ACT35, CD134, or TNFRSF4) belongs to the TNFR superfamily and is a type I transmembrane protein. OX40 is primarily expressed on activated CD4+ and CD8+ T cells, while its expression levels on NK and NKT cells are relatively low. OX40L (also known as CD252, TNFSF4, CD134L, or GP34) is the ligand for OX40 and is a type II glycoprotein. OX40L is predominantly expressed on antigen-presenting cells, such as B cells and dendritic cells.

Similar to other members of the TNF family, OX40 signaling is mediated via TNF receptor-associated factors (TRAFs). In vivo, OX40 signals are transduced into T cells through TRAF2 and TRAF5; in vitro, this transduction occurs via TRAF1, TRAF3, and TRAF5. The expression of OX40 and OX40L is regulated by a variety of factors. On T cells, OX40 expression is induced by signals such as those from the TCR, CD28/CD80, and CD40/CD40L pathways, reaching peak expression levels 48 to 72 hours after T cell activation. While TCR signaling can initiate OX40 expression on various cell types, CD28 and other cytokines can further enhance its expression on activated T cells. IL-2, IL-4, and TNF can augment or prolong OX40 expression. The OX40/OX40L axis plays a pivotal role in enhancing effector T cell function, maintaining effector T cell survival, and inhibiting effector T cell apoptosis.

HEK293 Human OX40/NFκB-Luc Cell Model—effectively simulates the signal transduction process of OX40 *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human OX40/NFκB-Luc Cell Model

HEK293 cell line expressing full length human OX40. Expression is confirmed by flow cytometry.

Figure 2. Recombinant OX40/NFκB-Luc/HEK293 stably expressing OX40.

Figure 3. Dose Response of OX40L in OX40 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 OX40/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.