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Activin A is an important member of the transforming growth factor-β (TGF-β) superfamily, a pleiotropic factor involved in the pathogenesis of immune dysregulatory diseases such as allergies, autoimmune disorders, and cancer.

  
Activin A signals through two types of type I receptors and two types of type II receptors, which assemble into the final receptor complex upon ligand binding. The type I receptors include activin receptor type 1A (or activin receptor-like kinase 2, ALK2), activin receptor type 1B (or ALK4), and activin receptor type 1C (or ALK7). Activin A has a preference for ALK4 binding and exhibits lower affinity for ALK2 and ALK7. The type II receptors are activin receptor type IIA (ActRIIA) and activin receptor type IIB (ActRIIB), characterized by constitutively active serine/threonine kinase activity. Once Activin A binds to its type II receptor, two type I receptors are recruited and phosphorylated by the type II receptor, an event that activates their kinase activity. Most TGF-β superfamily members bind to ActRIIB or ActRIIA, while only a few ligands bind with high affinity to ALK4. In addition to the canonical receptor serine/threonine kinase-Smad pathway, Activin A has been shown to participate in pathways such as P38, ERK, PI3K, and WNT.

The ACVR2B Effector Reporter Cell model effectively mimics the signal transduction process of ACVR2A in vivo, as illustrated in Figure.

Figure 1. Schematic of the ACVR2B Effector Reporter Cell Model.

Figure 2.  ACVR2B Effector Reporter Cell sequencing data, ACVR2A(NM_001616.5): c.409_410insT/ ACVR2A:p.Y137Lfs*76.

Figure 3. Dose Response of Recombinant Human Activin A  in ACVR2B Effector Cell(2C8).Dose Response of Recombinant Human GDF-8 in ACVR2B Effector Reporter Cell(2C8).Dose Response of Recombinant Human GDF-11 in ACVR2B Effector Reporter Cell (2C8).

Figure 4. Inhibition of Human GDF-8 induced Reporter Activity byACVR2 Blocking Ab in ACVR2B Effector Reporter Cell(2C8). Inhibition of Human GDF-11 induced Reporter Activity by ACVR2 Blocking Ab in ACVR2B Effector Reporter Cell(2C8).

Figure 5. Inhibition of  Recombinant Human Activin A-induced Reporter Activity by Samples in ACVR2B Effector Reporter Cell (2C8).

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 ACVR2B 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.