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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 being upregulated and trimerized by 4-1BBL on T cells, 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 signaling—mediated via TRAF1 and TRAF2—thereby activating the NF-κB, AKT, p38 MAPK, and ERK pathways. NF-κB induces 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 HEK293 Human 41BB/NFκB-Luc Cell model effectively mimics the in vivo 41BB signaling transduction process. The principle is illustrated in the figure below.

Figure 1. Schematic diagram of the HEK293 Human 41BB/NFκB-Luc Cell model.

Figure 2. Recombinant 41BB/NFκB-Luc/HEK293 stably expressing 41BB.

Figure 3. Dose Response of 4-1BBL in 4-1BB NF-κB-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 41BB/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.