Camp enzyme immunoassay eia kit

Ginsenoside Ro (G-Ro, Figure 1) was obtained from Ambo Institute (Daejeon, Korea). Thrombin was purchased from Chrono-Log Corporation (Havertown, PA, USA). ATP assay kit was purchased from Biomedical Research Service Center (Buffalo, NY, USA). Serotonin ELISA kit was purchased from Labor Diagnostika Nord GmbH & Corporation (Nordhorn, Germany). A-kinase inhibitor Rp-8-Br-cAMPS, G-kinase inhibitor Rp-8-Br-cGMPS, and 2-acetoxymethyl (Fura 2-AM) were obtained from Sigma Chemical Corporation (St. Louis, MO, USA). Lactate dehydrogenase (LDH) cytotoxicity assay kit, cAMP enzyme immunoassay (EIA) kit, and cGMP enzyme immunoassay (EIA) kit were obtained from Cayman Chemical (Ann Arbor, MI, USA). Anti-IP₃-receptor type I, antiphosphor-IP₃-receptor type I (Ser¹⁷⁵⁶), anti-ERK (1/2), antiphosphor-ERK (1/2), anti-rabbit IgG-horseradish peroxidase conjugate (HRP), and lysis buffer were obtained from Cell Signaling (Beverly, MA, USA). Anti-β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal to CD62P (p-selectin) antibody was purchased from Biolegend (San Diego, CA, USA). Polyvinylidene difluoride (PVDF) membrane was from GE Healthcare (Piscataway, New Jersey, USA). Enhanced chemiluminescence solution (ECL) was from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK).

Enzyme-linked immunoassays for cAMP were performed using a cAMP enzyme immunoassay (EIA) kit (Cayman Chemicals, Grand Rapids, MI, USA) following the manufacturer's instructions. In brief, C57/BL6 mice were anaesthetized, and 2 μl of 10⁻¹⁰ M PACAP was applied to both ocular surfaces. Both infraorbital lacrimal glands were removed at 0, 7.5, 15 or 30 min after the application of the eye drops (n=8). They were then homogenized in 200 μl of 5% trifluoroacetic acid on ice. After centrifugation at 1,500g for 10 min, the lysate was mixed with 1 ml of ether for 10 s. After removal of the ether, the aqueous layer was acetylated according to the EIA kit manufacturer's instructions and used for the EIA assay. The absorption in each well was measured with a plate reader (POLARstar Omega; BMG LABTECH GmbH, Offenburg, Germany).