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HEK293 Human CD40/NFκB-Luc Cell reporter gene target model effectively mimics the in vivo CD40 signaling transduction process. The principle is illustrated in the figure below.

Figure 1. Schematic diagram of the HEK293 Human CD40/NFκB-Luc Cell model.

Figure 2. Recombinant HEK293 Human CD40/NFκB-Luc Cell constitutively expressing CD40.

Figure 3. Dose Response of Human CD40L in CD40 NFκB-Luc HEK293( C4).

Figure 4. Blocking of hCD40L induced Activity by CD40L Blocking Ab in CD40 NFκB-Luc HEK293(C4). Blocking of CD40L induced Activity by CD40L Blocking Ab in CD40 NFκB-Luc HEK293 Cell(C4) with CD40L Target Cell(C7).

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 CD40/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.