Camp assay cell lysis buffer

The medium of cultured parietal cells was replaced with DMEM/F-12 medium containing 10 μM RO-201724 (Sigma-Aldrich Co.) prior to treatment with test materials. The parietal cells were pretreated with C. tricuspidata extracts or 10 μM of ranitidine hydrochloride for 5 min, followed by 10 μM of the H2R specific agonist dimaprit (Sigma-Aldrich Co.) for 30 min. After 30 min of dimaprit treatment, parietal cells were treated with cAMP assay cell lysis buffer (R&D Systems) and immediately frozen. The frozen cells were disrupted by repeatedly freezing and thawing three times, centrifuged at 800 g for 15 min, and cAMP production was measured in the supernatant. cAMP assays were performed using the Rat/Mouse cAMP assay kit (R&D Systems).

Gastric mucosa isolated from rats was washed twice with phosphate buffer saline (PBS) and collected by centrifugation at 800 g. The gastric mucosa was treated with cAMP assay cell lysis buffer (R&D Systems, Minneapolis, MN, USA) and ground using a tissue homogenizer. The gastric mucosa suspension was centrifuged at 2,000 g and the supernatant was used to measure cAMP levels using a rat/mouse cAMP assay kit (R&D Systems).

U937 cells were cultured in RPMI-1640 medium, centrifuged at 200 g, and suspended in RPMI-1640 medium containing 10 μM RO-20-1724 (Sigma, St. Louis, MO, USA). Cell suspensions were pretreated with C. tricuspidata extracts or 10 μM of ranitidine hydrochloride (Sigma) for 5 min and then treated with 10 μM of H₂R specific agonist dimaprit (Sigma) for 20 min. After 20 min of dimaprit treatment, U937 cells recovered by centrifugation (1,800 g) were treated with cAMP assay cell lysis buffer (R&D Systems, Minneapolis, MN, USA) and immediately frozen. The frozen cells were disrupted by repeatedly freezing and thawing three times, centrifuged at 2,000 g for 15 min, and cAMP production was measured in the supernatant. The cAMP assay was performed using a cAMP assay kit from R&D Systems.