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The cAMP response element-binding protein (CREB) is a transcription factor located within the cell nucleus. Upon phosphorylation at the Ser133 site by various receptor-activated protein kinases—such as protein kinase A (PKA), calmodulin-dependent protein kinase (CaMK), mitogen-activated protein kinase (MAPK), and others—it binds to the cAMP response element (CRE) located within the promoters of target genes. The CRE serves as the primary binding site for CREB and is responsible for its activation. The CRE acts as a focal point for numerous extracellular and intracellular signaling pathways, including those involving cAMP, calcium, GPCRs (G protein-coupled receptors), and neurotrophic factors. The cAMP/PKA signaling pathway is critical for a wide range of biological processes and organisms. Within the cAMP/PKA signaling pathway, CREB is activated through phosphorylation by PKA and subsequently binds to the CRE, which typically features the consensus motif 5'-TGACGTCA-3'. Given that the CRE functions as a regulator of the cAMP/PKA signaling pathway, it serves as a valuable target for investigating the effects of various inhibitors.

CRE-Luc HEK293 reporter cells are HEK293 cells in which the Luc luciferase reporter gene is regulated by CRE. Forskolin is a naturally occurring direct activator of the adenylate cyclase (AC)/cAMP/CREB pathway; the underlying mechanism is illustrated in the figure below.

Figure 1: Schematic Diagram of CRE-Luc HEK293

Figure 2. Detect Luciferase assay by Ultra Luciferase Detection Kit RQH0001（we strongly suggest to purchase from Reqbio). Induction of CRE activity by Forskolin in CRE-Luc HEK293(C5).

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 CRE-Luc Reporter 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.