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Angiopoietins (ANG) are key secreted proteins that regulate vascular stability, comprising two core subtypes: ANG1 and ANG2. ANG1 is secreted by pericytes and maintains vascular integrity and maturity by activating the TIE2 receptor. ANG2 is secreted in an autocrine manner by stressed endothelial cells; acting as a switch for vascular destabilization, it induces vascular regression in low-VEGF environments (e.g., diabetic retinopathy) while promoting pathological angiogenesis in high-VEGF environments (e.g., tumor angiogenesis). Its expression is significantly induced by hypoxia (via HIF-1α) and inflammatory cytokines (via TNF-α).

TIE2 (a tyrosine kinase receptor) is primarily expressed on the surface of vascular endothelial cells. Its extracellular domain contains immunoglobulin-like domains (which bind to ANG) and EGF-like domains (which mediate dimerization), while its intracellular domain houses tyrosine kinase active sites (at Y1100/Y1108). Upon ligand activation, TIE2 regulates endothelial cell survival, migration, and vascular barrier function via the PI3K/AKT and MAPK signaling pathways. Under pathological conditions (such as sepsis or cancer), aberrant TIE2 signaling leads to vascular leakage or abnormal proliferation.

The ANG/TIE2 Effector Reporter Cell model accurately simulates the in vivo signal transduction processes of the ANG/TIE2 pathway; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human ANG/TIE2 Effector Reporter Cell Model

Figure 2. Recombinant ANG/TIE2 Effector Reporter Cell stably expressing TIE2.

Figure 3. Dose Response of Ligands in ANG/TIE2 Effector Reporter Cell( C21).

Figure 4. Inhibition of Human ANG1-induced Reporter Activity by Trebananib in ANG/TIE2 Effector Reporter Cell( C21). Inhibition of Human ANG2-induced Reporter Activity by Samples in ANG/TIE2 Effector Reporter Cell(C21).

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 ANG/TIE2 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.