Camp enzyme immunoassay kit

R122H and V207A HA-tagged human P2Y₁₂ constructs were generated by site-directed mutagenesis (Eurofins MWG Operon, Ebersberg, Germany) and were transfected into either HEK293 or 1321N1 cells according to previously described methods (17 ). Cell surface P2Y₁₂ expression in the transfected cells was determined by enzyme linked immunosorbent assay (ELISA) and by immunofluorescence microscopy using murine anti-HA antibody (HA-11) as described previously (17 ). P2Y₁₂ receptor function was measured by incubating the transfected cells with 1 µM forsoklin (Sigma-Aldrich, Gillingham, UK) to increase basal cAMP levels. The cells were then incubated with 50 µM-10 nM ADP before residual cAMP concentrations were determined in cell lysates by ELISA (Sigma-Aldrich cAMP Enzyme Immunoassay Kit, Gillingham, UK).

The cAMP concentration was determined according to the previous work with modification [32 (link)]. M. purpureus HJ11 strains were cultivated in GM medium at 30 °C. Fresh mycelia were harvested, frozen and lyophilized and ground into powder using liquid nitrogen. The powder sample was kept in chilled 6% (w/v) trichloro acetic acid (TCA) solution and incubated for for 20 min. After centrifugation of the mixture at 3500×g at 4 °C for 20 min, the supernatant was extracted 3 times with 10 volumes of diethyl ether to remove TCA residues. The resulting extract was dried before further analysis. The cAMP levels were quantified using cAMP Enzyme Immunoassay Kit, Direct (Sigma-Aldrich, St. Louis, MO) following the manufacturer's instructions. In total, assay was repeated three times independently with three biological replicates for each strain.

Cells were stimulated for 15 min with dDAVP (1 nM or 1 μM, Sigma) or 25 μM forskolin (Sigma) dissolved in DMSO in the presence of 3-isobutyl-1-methylxanthine (IBMX). Controls were DMSO + IBMX. Total cAMP levels were measured using a commercially available cAMP enzyme immunoassay kit (Sigma).

Translocation of Cu-Zn SOD–CyaA into host cells was assayed using the CyaA fusion approach. After infection of J774.A1 cells (moi 100:1) for the indicated times in 96-well plates (10⁵ cells per well), cells were gently washed five times with PBS and lysed. Intracellular cAMP levels were determined by cAMP enzyme immunoassay kit from Sigma-Aldrich Corporation LLC as described by the manufacturer [9 (link)].

Chemicals used in this study include atropine sulfate, carbachol (CCh), dicyclomine, isoprenaline, verapamil, and papaverine were procured from Sigma (St. Louis, MO, United States). The cAMP enzyme immunoassay kit used to estimate cAMP was procured from Sigma-Aldrich Co., United States. It is worth noting that all other chemicals used were of analytical grade.
Guinea-pigs (either sex, weighing 510–560 g) were housed at the animal house located at College of Pharmacy, PSAU, Saudi Arabia in a controlled environment maintained at 25 ± 2°C. The animals were provided with free access to water and commercial standard diet. All experiments were done as per the guidelines of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (National Research Council, 1996 ). The study was approved by Bio-Ethical Research Committee (BERC), Prince Sattam Bin Abdulaziz University (Approval number BERC-004–12–19).