Catchpoint camp fluorescent assay kit

Prm1-LAPD (1 × 10⁶) sperm were illuminated with 690 nm light for 5 min. Afterwards, sperm were quenched with 0.5 M HClO₄, vortexed, frozen in liquid N₂, thawed, and neutralized by addition of K₃PO₄ (0.24 M final concentration). The salt precipitate and cell debris were sedimented by centrifugation (15 min, 20,000× g, 4 °C). The cAMP content in the supernatant was determined by a competitive immunoassay (CatchPoint cAMP Fluorescent Assay Kit, Molecular Devices, San Jose, USA) including an acetylation step for higher sensitivity. Calibration curves were obtained by serial dilutions of cAMP standards. 96-well plates were analyzed by using a microplate reader (FLUOstar Omega; BMGLabtech, Ortenberg, Germany).

NIH3T3-(Opsin/GFP) and NIH3T3-(GFP) were plated in two 96-well plates at a density of 50,000 cells per well in 100 µL of DMEM medium containing 10% FBS and antibiotics. After 24 h, cells were washed with Krebs Ringer bicarbonate buffer containing glucose (KRBG), and incubated with KRBG buffer containing 100 µM cAMP specific phosphodiesterase inhibitor, Ro 20–1724 (Tocris, UK) at RT for 10 min. Under dim red light, cells then were treated with 25 µL of 6× its final concentration of forskolin (final 20 µM) followed by addition of 25 µL 6× final concentration of 9-cis-retinal, YC-001 or 9-cis-retinal and YC-001 together. While one plate then was wrapped with aluminum foil, the second plate was exposed to regular room light. Both plates were kept in a cell culture incubator for 15 min at 37 °C in 5% CO₂. Levels of accumulated cAMP were detected with the Catchpoint cAMP fluorescent assay kit (Molecular Devices, Sunnyvale, CA USA) and the fluorescence with excitation/emission at 530/590 nm was read with a Flexstation3 plate reader (Molecular Devices) as described in the manufacturer’s protocol.