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Glucocorticoids (GCs) are steroid hormones that exert significant influence on numerous physiological functions through their diverse roles in inflammation, growth, metabolism, and development. Owing to their anti-proliferative and anti-inflammatory properties, GCs have been utilized for decades in the treatment of malignant tumors, as well as various inflammatory and autoimmune diseases. Clinically employed synthetic GCs include dexamethasone (Dex), betamethasone, cortisone, hydrocortisone, methylprednisolone, and prednisolone.

Endogenous GCs, produced by the adrenal cortex, are under the strict regulation of the HPA (hypothalamic-pituitary-adrenal) axis; their lipophilic nature enables them to readily diffuse across cell membranes. Intracellularly, GCs mediate their effects via the glucocorticoid receptor (GR)—a transcription factor belonging to the nuclear hormone receptor superfamily. In the absence of GCs, the GR forms a complex with chaperone proteins and remains sequestered within the cytoplasm; while residing in the cytoplasm, these chaperone proteins maintain the receptor in an inactive state and organize its structural conformation to facilitate ligand binding. Upon ligand binding, the GR becomes activated and dynamically shuttles between the cytoplasm and the nucleus, where it functions as a transcription factor to positively and negatively regulate numerous GR-dependent genes.

Transcriptional induction by the GR is primarily triggered by the binding of GR homodimers to promoter regions containing glucocorticoid response elements (GREs)—a mechanism termed GR-dependent transactivation (TA). Conversely, GR-dependent transrepression (TR) is mediated by the interaction of the GR with DNA-binding transcription factors—such as NF-κB and Activator Protein-1 (AP-1)—resulting in the suppression of their respective inflammatory signaling cascades. Elevated levels of the GR have been observed in certain patients suffering from glucocorticoid-resistant asthma, rheumatoid arthritis, ulcerative colitis, systemic lupus erythematosus, acute lymphocytic leukemia, or chronic lymphocytic leukemia. II. Description
HEK293 cells regulated by GR-GAL4 and expressing the Luc luciferase reporter gene. Dexamethasone (Hexadecadrol) is an agonist of the Glucocorticoid Receptor and an apoptosis inducer; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the GR-GAL4 Luciferase Reporter HEK293 Cell Mechanism

Figure 2. Dose Response of Dexamethasone in GR-GAL4 Luciferase Reporter HEK293 (C12).

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 GR-GAL4 Luciferase 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.