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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.

The Jurkat E6.1 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 theJurkat E6.1 Human OX40/NFκB-Luc Cell Model

Jurkat E6.1 cell line expressing full length human OX40. Expression is confirmed by flow cytometry.

Figure 2. Recombinant OX40/NFκB-Luc/Jurkat stably expressing human OX40.

Figure 3. Dose response of OX40L in OX40 NFκB-Luc Jurkat(C29).

Figure 4. Dose Response of OX40 Agonist Antibody in OX40 NFκB-Luc Jurkat(C29) with FCGR2B(FcγRIIb) CHO.

Figure 5. Blocking of OX40L Protein induced Activity in OX40 NFκB-Luc Jurkat Cell(C29) by OX40L Blocking Ab. Blocking of OX40L induced OX40 NFκB-Luc Jurkat(C29) Activity by OX40L Blocking Ab with OX40L CHO.

Cell Passage Procedures

1.This cell line grows in suspension.
2.Upon receipt, cells should be thawed immediately or stored in liquid nitrogen until use.
3.Before thawing, pre-warm the water bath and culture medium to 37 °C, and prepare a small amount of dry ice.
4.Remove the cryovial from storage and transport it to the cell culture laboratory on dry ice.
5.Rapidly thaw the cells in a 37 °C water bath. Once the cells are completely thawed, spray the cryovial with 70% ethanol for disinfection and transfer it to a biosafety cabinet.
6.Add 10 mL of pre-warmed culture medium into a 15 mL centrifuge tube. Transfer the contents of the cryovial into the tube and centrifuge at 1000 rpm for 5 minutes.
7.Carefully discard the supernatant. Resuspend the cell pellet in 5 mL of pre-warmed culture medium by gentle pipetting. Immediately perform cell counting and adjust the cell density to 3–6 × 10⁵ cells/mL based on the counting results, then transfer the cells into a culture flask.
8.Count the cells every 1–2 days. When the cell density exceeds 1 × 10⁶ cells/mL, passage the cells promptly or add fresh culture medium. Maintain the cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL.

Suspension Cell Cryopreservation Procedure:

1.Collect 8 × 10⁶ cells, centrifuge, and discard the supernatant.
2.Add 1 mL of cell freezing medium (90% FBS + 10% DMSO) and gently pipette to mix thoroughly. Transfer the suspension into a cryovial.
3.Immediately place the cryovial into a controlled-rate freezing container (Nalgene 5100-0001), fill with isopropanol up to the indicated level, and store at −80 °C.
4.After 24 hours, transfer the cryovial to liquid nitrogen for long-term storage.