Pka inhibitor fragment 6 22 amide

Primary microglia cultures were prepared from P0 mouse pups following a published protocol (Lian et al, 2016). Briefly, the cortices of the brains were harvested and digested by trypsin. The cells were plated in poly‐d‐lysine (PDL)‐coated T‐75 flasks as mixed glial cultures and let grow till confluent astrocyte layers form. The microglia were isolated by tapping the flasks vigorously. Purified microglia were seeded into PDL‐coated 96‐well plates at 20,000 cells/well density and let recover overnight. The cells were treated with 10 pg of SEC‐purified oAβ, 100 μM isoproterenol, 50 μM cAMP‐Rp (Tocris 1337), 100 nM KT5720 (Tocris 1288), and 100 nM PKA inhibitor fragment (6‐22) amide (Tocris 1904). Cells were harvested after 4 h of incubation at 37°C and lysed for RNA analysis.

Drugs were bath-applied to the whole slice through the perfusion system by dilution of concentrated stock solutions (prepared in water or DMSO) in ACSF, or by adding the drugs to the patch pipette solution when it was applied intracellularly to the postsynaptic cell only. If the drug was not water-soluble, vehicle control experiments were carried out. For each set of recordings, interleaved control and drug conditions were carried out and were pseudorandomly chosen. The following drugs were used in this study: 100 μM dopamine hydrochloride (Sigma–Aldrich, Dorset, United Kingdom), 100 μM D-AP5 (Tocris Bioscience, Bristol, United Kingdom), 10 µM nimodipine (Tocris Bioscience), 1 µM PKA inhibitor fragment (6-22) amide (Tocris Bioscience), 0.5 mM anisomycin (stock solution in EtOH; Tocris Bioscience), and 1 mM MK801 (Tocris Bioscience).