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The natriuretic peptide (NP) family, consisting of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), D-type natriuretic peptide (DNP), and urodilatin (UNP), plays an indispensable role in hypertension and cardiovascular regulation. ANP and BNP are effective endogenous hypotensive hormones with diuretic, natriuretic, vasodilatory, anti-proliferative, anti-inflammatory, and anti-hypertrophic effects, which can reduce and control fluid volume, blood pressure, and cardiovascular diseases.

  
Different NP receptors are categorized as: NP receptor a (NPRA, NPR1), NP receptor b (NPRB, NPR2), and NP receptor c (NPRC, NPR3). ANP and BNP bind and activate NPR1, which generates the intracellular second messenger cGMP after hormone binding. CNP activates NPR2 and produces cGMP. All three NPs (ANP, BNP, CNP) bind to NPR3, which lacks a guanylyl cyclase catalytic domain and does not increase intracellular cGMP levels.

  
When bound to ANP or BNP, NPR1 transmits signals by catalytically synthesizing cyclic guanosine monophosphate (cGMP). Elevated intracellular cGMP levels stimulate cGMP-dependent phosphodiesterases, cGMP-dependent protein kinases (PKG-I and -II), and cGMP-dependent ion channels.

The BNP/NPR1 Effector Reporter Cell model effectively mimics the intracellular signaling process of NPR1 in vivo, with the principle illustrated in the figure below.

Figure 1. Schematic of the BNP/NPR1 Effector Reporter Cell Model.

Figure 2. WB of BNP/NPR1 Effector Reporter Cell.

Figure 3. Dose Response of Recombinant hBNP in NPR1 Effector Reporter Cells (C4).

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 BNP/NPR1 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.