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G-CSF (Granulocyte Colony-Stimulating Factor) is a cytokine naturally produced by the human body. Its primary function is to direct the bone marrow—acting as a "factory"—to produce and release neutrophils (a critical type of white blood cell), serving as a central commander within the immune system for combating infections. Its receptor, G-CSFR (Granulocyte Colony-Stimulating Factor Receptor), is widely distributed on the surface of hematopoietic stem cells and neutrophils. When G-CSF binds to G-CSFR—much like a key fitting into a lock—it triggers intracellular signaling pathways that promote the proliferation and differentiation of hematopoietic stem cells into mature neutrophils, while also enhancing their functional activity.

The HEK293 Human G-CSF/G-CSFR Effector Reporter Cell model effectively mimics the in vivo G-CSF/G-CSFR signaling pathway.

Figure 1. 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.