Enzyme immunoassay kit

The mice were injected daily with saline or PF (1 mg/kg) for 7 consecutive days. The blood samples were obtained from the orbit and transferred immediately into tubes with 50 mM EDTA, which was followed by centrifuging at 3,000 g for 10 min at 4°C. Fifty microliters of plasma were separated into tubes containing 1 μl 100 mM PMSF and 2.5 μl 1 N HCl, and they were stored at –80°C. The collections were staggered to match the controls with the treated samples. The plasma levels of total ghrelin (including ghrelin and desacyl-ghrelin) and ghrelin were measured using the enzyme immunoassay kits (catalog# EZRGRT–91K, catalog# EZRGRA–90K, Millipore, Hayward, CA, United States).

The frozen kidneys were ground to fine powder under liquid nitrogen in a stainless steel mortar. After the liquid nitrogen had evaporated, tissue was weighed and homogenized in 10 volumes of 0.1 M HCl. After centrifugation at 600 g for 10 min at room temperature, the supernatants were collected and assayed for cAMP without acetylation using an enzyme immunoassay kit (Sigma-Aldrich, Inc., St. Louis, MO, USA). The protein content was determined by using the BCA protein assay kit from Pierce (Rockford, Illinois, USA). The results were expressed in pmol/mg of protein.

The intracellular cyclic-AMP analysis was performed according to a method described elsewhere56 (link), with slight modifications. Briefly, B16 mouse melanoma cells stably expressing human melanocortin receptor MC1R were grown to confluence in DMEM medium (Sigma-Aldrich, USA) containing 10% fetal calf serum (FCS) and 1x anti-anti (Gibco). The cells were seeded in 12-well plates 48 h before assay and grown to 10⁵ cells/well. For the assay, the medium was removed and cells were washed with 1 mL of media. Then 1 mL DMEM media (without FCS) containing 100 μM 3-isobutyl-1-methylxanthine (IBMX) was added to each well and incubated for 30 min at 37 °C. The media was then discarded and various concentrations of α-MSH and KKK-MSH viz., 600 nM, 1 μM and 10 μM, in 1 mL fresh 25% DMEM media (without FCS) in PBS containing 100 μM IBMX were added to the appropriate wells and incubated for 10 min at 37 °C, 5% CO₂. After 10 min the reaction was stopped by aspirating the media and washing the wells with 1 mL PBS. 200 μL 0.1 M hydrochloric acid was then added to each well and the wells were scrapped. The lysates were centrifuged at 600 rcf for 5 min and the supernatants were collected and diluted 1000 times in 0.1 M HCl and used for analysis of the total cyclic-AMP content using Enzyme Immunoassay Kit (Sigma-Aldrich, USA, CA200) according to the manufacturer’s instructions.

cAMP measurements were performed using the cAMP Enzyme Immunoassay kit (CA-200, Sigma-Aldrich, St Louis, MO, USA), following the acetylated version of protocol supplied by the vendor. Cells were plated in 96-well plates at 9,000 cells per well and allowed to attach in complete medium at 33 °C and 5% CO₂ for 24 hr. Cells were treated with 10 μl DMSO or compounds and incubated in complete medium at 33 °C and 5% CO₂ for 15, 30, and 120 minutes at which time the medium was removed and the cells were lysed with 250 μl of 0.1 N HCl for 20 minutes. Equivalent amounts of samples were used in the ELISA assay as determined by protein concentration. Protein concentrations were determined using the Bradford protein assay (Bio Rad).

Serum and urine cGMP were measured using enzyme immunoassay kit (#CG201-1KT, Sigma-Aldrich). Creatinine was measured using Quantichrome creatinine assay kit (#DICT-500, Bioassay systems) respectively. The urinary albumin/creatinine ratio was measured by using the commercial kits Albuwell (#1011, Exocell).