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Erythropoietin (EPO) is a glycoprotein hormone secreted by the kidneys (in adults) or the liver (in fetuses), whose primary function is to regulate red blood cell production. EPO belongs to the Type I cytokine superfamily and consists of 165 amino acids arranged into four α-helices. In humans, due to high levels of glycosylation, the plasma half-life of renally produced EPO is 5–6 hours. When red blood cell levels decline, renal tubulointerstitial cells detect relative hypoxia and secrete EPO into the circulation via a classical endocrine mechanism. EPO then migrates to the bone marrow, where it binds to the dimeric erythropoietin receptor (EPOR) on erythroid progenitor cells, thereby promoting erythropoiesis.

      EPOR belongs to the type I cytokine receptor superfamily and is a single transmembrane protein composed of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signaling domain. Upon binding to EPOR, EPO induces receptor dimerization, triggering intracellular JAK2 autophosphorylation and activating downstream proliferation, differentiation, and anti-apoptotic signaling pathways.

The EPO Effector Reporter Cell model effectively mimics the in vivo signal transduction process of EPO and EPOR; the underlying mechanism is illustrated in the figure below.

Figure 1. Schematic diagram of the EPO Effector Reporter Cell model

Figure 2. Recombinant hEPO Effector Reporter Cell constitutively expressing EPOR.

Figure 3. Dose Response of Recombinant Human EPO in EPO Effector Reporter Cell (C5).

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 EPO 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.