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4-1BB [also known as CD137 or Tumor Necrosis Factor Receptor Superfamily Member 9 (TNFRSF9)] is a glycosylated Type I transmembrane protein belonging to the TNFR superfamily; it consists of four extracellular cysteine-rich pseudo-repeat domains. The cytoplasmic region of 4-1BB contains binding motifs for Tumor Necrosis Factor Receptor-Associated Factors (TRAFs). 4-1BB expression is primarily found on the surface of activated cytotoxic CD8+ T cells and helper CD4+ T cells; following activation, 4-1BB expression can also be induced in NK cells, B cells, monocytes, and dendritic cells. As an inducible co-stimulatory molecule, 4-1BB enhances T-cell responses to antigens. Upon upregulation and trimerization on T cells by its ligand, 4-1BBL, 4-1BB recruits TRAF adaptor proteins to the cytoplasmic TRAF-binding motifs, thereby initiating co-stimulatory signaling.

4-1BBL (TNFSF9) is the primary ligand for 4-1BB; it is predominantly expressed in APCs, B cells, macrophages, and other cell types. Upon binding to 4-1BB, it induces downstream signaling—mediated via TRAF1 and TRAF2—thereby activating the NF-κB, AKT, p38 MAPK, and ERK pathways. NF-κB drives the transcription of BCL-XL, BCL-2, and c-FLIP, and stimulates the expression of IL-2, IL-4, and IFN-γ, thereby promoting T-cell survival and cytotoxic activity. Consequently, the 4-1BB/4-1BBL signaling pathway can promote the survival of various cell types.

The Jurkat E6.1 Human 41BB/NFκB-Luc Reporter Cell effectively simulates the 4-1BB signal transduction process in vivo, as shown in the figure below.

Figure 1. Schematic diagram of the Jurkat E6.1 Human 41BB/NFκB-Luc Cell Model

Figure 2. Recombinant 41BB/NFκB-Luc/Jurkat constitutively expressing 41BB.

Figure 3. Detect Luciferase assay by Ultra Luciferase Detection Kit RQPH0001（we strongly suggest to purchase from Reqbio). Dose Response of 4-1BBL in 4-1BB NFκB-Luc Jurkat (C43).

Figure 5. Blocking of 41BBL Protein induced 41BB NFκB-Luc Jurkat Cell (C43) Activity by 41BBL Blocking Ab. Blocking of 41BBL induced 41BB NFκB-Luc Jurkat Cell(C43) Activity by 41BBL Blocking Ab with 41BBL 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.