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DLL4 (Delta-like ligand 4) is primarily expressed in vascular endothelial cells and regulates vascular branching through a "tip cell–stalk cell" differentiation mechanism: DLL4 activates Notch1 signaling in adjacent cells, thereby inhibiting their differentiation into tip cells; this limits excessive vascular growth and maintains the structural order of the vascular network. Within the tumor microenvironment, DLL4 is highly expressed in abnormal vasculature, inducing structural vascular dysfunction (characterized by leakage and hypoperfusion) and promoting both hypoxia and chemotherapy resistance. Furthermore, DLL4 plays a role in neurodevelopment and T-cell differentiation; mutations in its gene can lead to congenital disorders such as Adams-Oliver syndrome.

As a core member of the Notch receptor family, Notch1 consists of an extracellular ligand-binding domain (containing EGF repeats), a transmembrane domain, and an intracellular domain (NICD). Upon the binding of DLL4 to the extracellular domain of Notch1, a two-step proteolytic cleavage process is triggered: ADAM proteases (e.g., ADAM10/17) cleave the extracellular segment, followed by the cleavage of the transmembrane region by γ-secretase, which releases the NICD into the cell nucleus. The NICD then forms a complex with the transcription factor CSL and the co-activator MAML, thereby activating downstream target genes (e.g., Hes1, Hey1, Myc) and regulating stem cell maintenance, cellular proliferation, and differentiation.

DLL4 and Notch1 are critical components of the Notch signaling pathway, regulating developmental processes, angiogenesis, and disease progression through cell-contact-dependent signaling. As a member of the Notch ligand family, DLL4 functions as a transmembrane protein that specifically binds to and activates the Notch1 receptor, thereby driving cell fate determination, tissue homeostasis, and pathological remodeling.

The DLL4/Notch1 Effector Reporter Cell model serves as an excellent *in vitro* surrogate for the *in vivo* DLL4/Notch1 signal transduction process; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the DLL4/Notch1 Effector Reporter Cell Model

Figure 2. Recombinant DLL4/Notch1 Effector Reporter Cell stably expressing Notch1.

Figure 3. Blocking of DLL4 induced DLL4/Notch1 Effector Reporter Cell Activity by DLL4 Neutralizing Antibody with DLL4 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 DLL4/Notch1 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.