Investigation of DNIC-Induced cGMP Formation

The MSC cells were plated on a cover glass (18 mm in diameter) loaded into a 24-well plate at a density of 1.7 × 10³ cells/well until the desired confluency was reached. After the culture media were removed, 100 μM of DNIC–COOH (or 2.5 μM of DNIC–COOMe) was added to the cell culture and incubated for 2 h. The culture media were removed before the MSC cells were washed three times with PBS, whereas the washed MSC cells were further fixed with 4% paraformaldehyde (PFA) solution for 15 min at room temperature. After the PFA solution was removed, the MSC cells were washed twice with PBS for 3 min and permeabilized using 0.1% Triton X-100 (in PBS) at room temperature for 4 min. Subsequently, the MSC cells were washed twice with PBS for 3 min and incubated with a blocking buffer (2.5% BSA in PBS) at room temperature for 30 min. Then, the MSC cells were incubated with primary antibody solution (1:1000 for rabbit anti-cGMP antibody, purchased from Biorbyt, Cambridge, U.K.) at room temperature for 1 h before the MSC cells were washed three times with PBS for 3 min. Cover glasses containing the fixed MSC cells were mounted onto a microscope slide loaded with Fluoroshield mounting medium with DAPI to stain the nuclei before the optical images were captured using a confocal imaging system (Leica TCS-SP-X AOBS).