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The insulin receptor (INSR) is a pivotal transmembrane receptor that regulates glucose metabolism, cell growth, and energy homeostasis. INSR consists of two extracellular α-subunits (ligand-binding domains) and two transmembrane β-subunits (kinase activity domains) linked by disulfide bonds to form an (α2β2) tetramer. Upon insulin binding to the α-subunits, the β-subunits undergo autophosphorylation, thereby activating their intrinsic tyrosine kinase activity; this, in turn, coordinates metabolic and proliferative functions via the PI3K-AKT and MAPK/ERK signaling pathways. Acting as a "molecular switch" for metabolic and proliferative signals, INSR occupies a central position in the pathogenesis of diabetes, cancer, and rare genetic disorders.

Through alternative splicing, INSR exists as two distinct isoforms: IRA and IRB. IRA serves as a receptor for insulin, proinsulin, and insulin-like growth factor 2 (IGF-2), mediating proliferative effects in embryonic and tumor tissues; conversely, IRB is more specialized for mediating insulin action within metabolic tissues, such as the liver, muscle, and adipose tissue. As a member of the tyrosine kinase receptor family, IRA possesses intrinsic tyrosine kinase activity within its intracellular domain; upon insulin binding, it triggers autophosphorylation and activates downstream signaling cascades.

The HEK293 Human IRA Effector Reporter Cell Model—effectively simulates the signal transduction process of Insulin *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of theHEK293 Human IRA Effector Reporter Cell Model

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

Figure 3. Dose Response of Recombinant Human Insulin in IRA Effeetor Reporter Cell (C11).

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 IRA 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.