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Fibroblast growth factor 21 (FGF21) is a multifunctional hormone-like protein and an important metabolic regulator. The Klotho family includes α-Klotho, β-Klotho, and γ-Klotho. The α-Klotho and β-Klotho genes encode type I transmembrane proteins; β-Klotho (KLB) is primarily expressed in the liver and adipose tissue and, upon binding to FGFR1c and FGFR4, it acts as a high-affinity receptor for FGF15/19 and FGF21, respectively, and regulates bile production and energy metabolism.

      The FGF21 receptor consists structurally of two components: FGFR and KLB. Both KLB and FGFR are essential, but neither alone is sufficient to support FGF21 signaling, as each protein has a unique role. KLB is a ligand-binding molecule capable of directly binding FGF21, thereby allowing the factor to activate FGFR, which acts as the active subunit. FGF21 binds to and activates a cell surface receptor complex composed of FGF receptors (FGFR1c/2c/3c) and the cofactor β-Klotho (KLB), leading to ERK1/2 phosphorylation. When bound, FGF21 stimulates insulin sensitivity and glucose metabolism, thereby leading to weight loss. Diseases associated with FGF21 include acquired lipodystrophy and hepatocellular clear cell carcinoma. Its associated pathways include lipoprotein metabolism and electron transport in the respiratory chain, ATP production via chemiosmotic coupling, and heat production via uncoupling. 

  The KLB Effector Reporter Cell model effectively mimics the in vivo KLB signaling pathway; the mechanism is illustrated in the figure below.

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

Figure 2. Dose Response of Recombinant Human FGF21 in KLB Effecttor Reporter Cell(C16).

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 KLB 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.