Cgmp enzyme immunoassay kit

The T84 cell assay was performed essentially as described previously [26 (link)]. Briefly, T84 cells (ATCC, Rockville, MD, USA) were seeded and grown to confluence on 48-well plates (Nunc, Roskilde, Denmark) in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) (Gibco life technologies, Paisley, UK), supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 0.2% gentamicin (LONZA, Walkersville, MD, USA). The cells were washed 3 times with 250 µL DMEM-F12 and pre-incubated with 40 µL DMEM-F12 containing 1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) for 10 min at 37 °C. A volume of 40 µL 1 µM native or mutant STh peptide was added to each well in duplicate (final peptide concentration 0.5 µM) and incubated for 30 min at 37 °C. Following incubation, the reaction medium was aspirated, and the cells were lysed with 0.1 M HCl at 20 °C for 20 min. Subsequently, the lysates were centrifuged at 16,000× g for 10 min, and the supernatants were collected to estimate cGMP levels using a cGMP enzyme immunoassay kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA). The analysis was conducted according to the manufacturer’s instructions.

The T84 cell assay was performed as described previously [15 (link)]. Briefly, T84 cells (ATCC, Rockville, MD, USA) were seeded and grown to confluence on Nunc 24-well plates (Thermo Fisher Scientific) in Gibco™ Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 ((DMEM/F-12) (Thermo Fisher Scientific)), supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 0.2% gentamicin (Lonza, Basel, Switzerland). Cells were washed thrice with 500 µL DMEM/-F-12, and pre-incubated with 200 µL DMEM/F-12 containing 1 mM 3-isobutyl-1-methylxanthine (Sigma–Aldrich) for 10 min at 37 °C. A volume of 200 µL of sample was added to each well and incubated for 60 min at 37 °C. Following incubation, the reaction medium was aspirated, and cells were lysed with 0.1 M HCl at 20 °C for 20 min. The lysates were centrifuged at 16,000× g for 10 min and supernatants were collected for analysis. Levels of cGMP were determined using a cGMP enzyme immunoassay kit (Enzo Life Sciences, Inc, Farmingdale, NY, USA) according to the manufacturer's instructions. STh purified from ETEC was used as a control.