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Cancer-associated inflammation is characterized by the presence of specific inflammatory cells and inflammatory mediators, including cytokines and chemokines. Recent evidence indicates that the Signal Transducer and Activator of Transcription (STAT) protein family—particularly STAT3—plays a pivotal role in the selective induction and maintenance of the pre-malignant inflammatory microenvironment during the initiation of malignant transformation and the progression of cancer.

Signal Transducer and Activator of Transcription 3 (STAT3) is a transcriptional activator that directly relays signals from extracellular receptors to the cell nucleus, mediating the expression of various genes and playing a significant role in cellular processes such as apoptosis. Upon stimulation by cytokines and growth factors, STAT3 undergoes phosphorylation by receptor-associated Janus kinases (JAKs); it then forms homodimers or heterodimers, translocates to the cell nucleus, and functions as a transcriptional activator. STAT3 signaling represents a major intrinsic pathway in malignant tumors, as it is frequently activated within malignant cells and plays a critical role in regulating numerous genes essential for cancer-associated inflammation within the tumor microenvironment.

STAT3-Luc HEK293 reporter cells are HEK293 cells engineered to express a Luciferase (Luc) reporter gene under the transcriptional control of STAT3. The binding of the cytokine IL-6 to its receptor complex initiates the JAK/STAT3 signaling pathway; the underlying mechanism is illustrated in the figure below.

Figure 1. Schematic Diagram of the STAT3-Luc HEK293 Principle

Figure 2. Dose Response of Human IL-6 Protein in STAT3-Luc HEK293 Cell(C7).

Figure 3. Inhibition of IL-6-induced Reporter Activity by IL-6 Neutralizing Antibody in STAT3-Luc HEK293(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 STAT3-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.