Cicaprost

For high-content screening (HCS) microscopy, hiPSCdN and rodent sensory neuron cultures were stimulated in 96-well imaging plates with 100 µL of maturation medium per well. Compounds used in this study were as follows: serotonin (5-HT, 10 mM in dH₂O) and fentanyl (10 mM in dH₂O) were purchased from Sigma-Aldrich; cicaprost (PGI2 analog, 10 mM stock in DMSO) was from Cayman Chemicals (Ann Arbor, MI); and forskolin (10 mM stock in DMSO), [D-Ala2]-deltorphin II (1 mM in dH₂O/0.1% BSA), dynorphin A (1 mM in dH₂O/0.1% BSA), [Met5]-enkephalin acetate salt (1 mM in dH₂O/0.1% BSA), and nociceptin (1 mM in dH₂O/0.1% BSA) were purchased from Tocris (Bristol, United Kingdom). All drugs were prepared as stocks and stored at −70°C or −20°C until use.
For stimulation, compounds were dissolved in 12.5 μL of PBS in 96-well V-bottom plates (NerbePlus, Winsen, Germany, 10-111-0000), prewarmed to 37°C, mixed with 50 μL of medium from the culture wells, and added back to the same wells. Stimulations were performed with automated 8-channel pipettes (Eppendorf, Hamburg, Germany) at low dispense speed on heated (37°C) blocks. The cells were fixed for 10 minutes at room temperature (RT) by adding 100 μL of 8% paraformaldehyde resulting in a final concentration of 4%.