Camp elisa kit

We randomly put eligible sheep COCs into the TCM-199 (Gibco) culture medium with or without Forskolin (100 μM) (F6886, Sigma) and IBMX (500 μM) (I5879, Sigma)—10 COCs in each group—and placed them into an in vitro culture in a 5% CO₂ incubator at 39 °C for 0 min, 10 min, and 1 h. The cultured sheep COCs were collected into a 1.5 mL centrifuge tube, to which 150 μL of 0.1 M HCl was added for lysis, and the sample supernatant was collected. An aliquot of each sample was assayed for cAMP levels using a cAMP ELISA kit (581001, Cayman, UK) according to the manufacturer’s protocol. A multifunctional enzyme plate analyzer (CYT-1000, GE HealThCare, Chicago, IL, USA) was used to detect OD values at 412 nm, draw the standard curve, and calculate the cAMP level in samples.

Freshly isolated neutrophils or bone marrow-derived macrophages (1 million cells) were treated with CGRP (1 nM) in RPMI with 10% FBS for 30 min at 37 °C with 5% CO₂. cAMP levels were quantified using a cAMP ELISA kit from Cayman Chemical (581001) according to the manufacturer’s instructions.

Differentiated brown adipocytes from different groups of mice were starved for 8 hrs, followed by treating the cells with or without 100 ng/mL IL18 and 20 µM H89 for 24 hrs. Cell were extraction with 0.1 M HCl and centrifuged at 1000 g for 10 min. cAMP level was determined using the cAMP ELISA Kit (581001, Cayman Chemical) according to manufacturers.

PRP (4 × 108 platelets/mL) was pre-incubated with different concentrations of lusianthridin at 37 °C for 5 min with continuous stirring. A positive control, the 3-isobutyl-1-methylxanthine (IBMX), is a phosphodiesterase inhibitor used to prevent cAMP catabolism. After that, ADP (4 µmol/L) was added to induce platelet aggregation and the reaction was terminated by 0.5% ice-cold ethanol. The samples were then vortexed, sonicated, and centrifuged to obtain the supernatant [33 (link)]. The cAMP ELISA kit (Cayman) was used to perform this assay and the experiments were carried out according to the manufacturer’s instructions. The developed yellow color was analyzed at the wavelength of 405–450 nm.

Intracellular cAMP concentrations of HepG2 and BEL7402 cells that were treated as described above were measured using a cAMP ELISA kit (Cayman Chemical, Ann Arbor, MI, USA). Briefly, 1 × 10⁵ cells were lysed in 0.1 M HCl for 20 minutes and then were scraped off and dissociated until the suspension was homogeneous. After centrifugation at 1000 g for 10 minutes, the cAMP concentration of each supernatant was measured according to the manufacturer's instruction. Briefly, 50 μL of each supernatant was added to 50 μL of cAMP AChE Tracer and 50 μL of cAMP ELISA antiserum in each well. After incubation at 4°C for 18 hours, the wells were rinsed, and 200 μL per well of Ellman's reagent was added. After incubation in the dark for 2 hours, the absorbance was measured at OD = 420 nm. The cAMP concentration of each sample was calculated according to the standard curve.

BmN cells were plated in 12-well tissue culture plates (2 × 10⁵ cells/well; Asahi glass, Yokohama, Japan) and maintained in normal growth medium (TC-100 [Applichem, Darmstadt, Germany], 10% FBS, and 50 μg/mL gentamycin). At 3 days post-seeding, the cells were pre-incubated in 800 μL of assay medium (TC-100 supplemented with 1 mM 1-methyl-3-isobutylxanthine [IBMX; Sigma-Aldrich, Tokyo, Japan]) and 1 mM phenylmethylsulfonyl fluoride for 10 min at 28°C. Following pre-incubation, 100 μL of a potential competitor and 100 μL of the peptide of interest were added sequentially to the medium and incubated at 28°C for 30 min. After incubation, the cells were washed twice with TC-100 and then immediately frozen in liquid nitrogen. Intracellular cAMP and cGMP were extracted by sonication with 300 μL 0.1 M HCl supplemented with 1 mM IBMX and then collected from the supernatant subsequent to centrifugation (4°C, 16,000 × g, 10 min). The extracted cAMP and cGMP were quantified in duplicate using a cAMP ELISA kit (Cayman Chemicals, Ann Arbor, MI, USA) and a cGMP EIA system (GE Healthcare, Hino, Japan), respectively. These experiments were repeated at least twice.