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G-CSF (Granulocyte Colony-Stimulating Factor), also known as CSF3, is a glycoprotein composed of approximately 174 amino acids. Primarily produced by monocytes, fibroblasts, vascular endothelial cells, and certain stromal cells, it plays a pivotal role in regulating inflammation and hematopoiesis. Within the cytokine superfamily, G-CSF belongs to the hematopoietic growth factor family—specifically the colony-stimulating factor family—which also includes M-CSF (macrophage colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), and Multi-CSF (i.e., IL-3).

CSF3R, also known as G-CSFR or CD114, is the specific cell-surface receptor for G-CSF. It belongs to the type I cytokine receptor family, a group also referred to as the hematopoietic factor receptor family or the erythropoietin receptor family. Upon binding to G-CSF, CSF3R undergoes homodimerization, which activates associated JAK-family tyrosine kinases (primarily JAK1 and JAK2). The activated JAKs phosphorylate specific tyrosine residues within the intracellular domain of CSF3R, creating docking sites for downstream signaling molecules and thereby initiating signaling pathways such as JAK-STAT, PI3K-AKT, or RAS-MAPK.

The G-CSF/G-CSFR Effector Reporter Cell  model effectively simulates the in vivo G-CSF signal transduction process, the principle is illustrated in the figure below.

Figure 1. Schematic diagram of the HEK293 Human G-CSF/G-CSFR Effector Reporter Cell model

Figure 2. Recombinant G-CSF/G-CSFR Effector Reporter Cell stably expressing G-CSFR.

Figure 3. Dose Response of Recombinant Human G-CSF in G-CSF/G-CSFR 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 G-CSF/G-CSFR 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.