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EGFR (Epidermal Growth Factor Receptor) is the receptor for epidermal growth factor (EGF) that mediates cell proliferation and signal transduction. EGFR belongs to the HER receptor family, which consists of four related proteins: EGFR (HER1/ErbB1), ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4). These are tyrosine kinase receptors located on the cell membrane surface and are activated by binding to ligands. EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and the inhibition of apoptosis.

      HER receptors are activated by binding to various ligands, including EGF, TGFA, heparin-binding EGF-like growth factor, dual-regulator, β-cell protein, and epithelial regulator. Upon binding of the ligand to the extracellular domain of the receptor, the receptor forms functionally active dimers [EGFR-EGFR (homodimer) or EGFR-HER2, EGFR-HER3, EGFR-HER4 (heterodimers)]. Dimerization induces activation of the tyrosine kinase domain, leading to autophosphorylation of the receptor at multiple tyrosine residues. This leads to the recruitment of a series of adaptor proteins (such as SHC and GRB2) and activates a series of intracellular signaling cascades that influence gene transcription, thereby leading to cancer cell proliferation, reduced apoptosis, invasion, and metastasis, and stimulating tumor-induced angiogenesis.

The EGFR Effector Reporter Cell model effectively mimics the in vivo EGFR signaling pathway; the underlying mechanism is illustrated in the figure below.

Figure 1. Schematic diagram of the EGFR Effector Reporter Cell model

Figure 2. Recombinant EGFR Effector Reporter Cell stably expressing EGFR.


Figure 3. Dose Response of  Recombinant Human EGF in EGFR Effector Reporter Cell (C9).



Figure 4. Inhibition of rhEGF-induced Reporter Activity by EGFR Blocking Ab in EGFR Effector Reporter Cell (C9). Inhibition of rhEGF-induced Reporter Activity by EGFR Inhibitor in EGFR Effector Reporter Cell (C9).

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 EGFR Effector 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.