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Epidermal growth factor (EGF) is a protein that stimulates cell growth and differentiation by binding to its receptor, EGFR. EGF signaling regulates various biological responses, such as proliferation, differentiation, cell movement, and survival. EGF binds to a specific, high-affinity, low-capacity receptor on the surface of responsive cells, known as the epidermal growth factor receptor (EGFR). EGFR is a member of the ErbB (erythroblastoma leukemia virus oncogene homolog) family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB1), Her2 (ErbB2), Her3 (ErbB3), and Her4 (ErbB4).

      Binding of EGF to the extracellular domain of EGFR leads to receptor dimerization, activation of intrinsic PTK (protein tyrosine kinase) activity, autophosphorylation of tyrosine, and the recruitment of various signaling proteins to the autophosphorylation site located primarily at the C-terminal tail of the receptor. Tyrosine phosphorylation of EGFR leads to the recruitment of various signaling proteins, including the adaptor proteins GRB2 and Nck, PLC-γ, SHC, STATs, and several other proteins and molecules.

      SREs are present in the promoters of various cellular genes involved in cell growth, differentiation, neuronal transmission, and muscle development, and are activated by serum, cytokines, tumor necrosis factor-α (TNF-α), hepatitis B virus-activated protein pX, proto-oncogenes, and extracellular stimuli. SREs serve as binding sites for the transcription factor serum response factor (SRF) and the three-component factor (TCF) complex. SRE is activated via two pathways: the TCF-dependent and RAS-dependent pathways (Ras-Raf-MEK-ERK signaling cascade), while the TCF-independent pathway (Rho-dependent pathway) involves the Rho family.

 SRE-Luc HEK293 reporter cells are HEK293 cells in which the SRE regulates the expression of the Luc luciferase reporter gene. The mechanism by which EGF binds to its receptor and activates SRE is shown in the figure below.

Figure 1. Schematic diagram of the SRE-Luc HEK293 cell model

Figure 2. Detect Luciferase assay by Ultra Luciferase Detection Kit CBPH0001（we strongly suggest to purchase from Cobioer). Dose Response of Recombinant Human EGF in SRE-Luc HEK293 (C5C16).

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 SRE-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.