Author: Clayton M. Carey; Sarah E. Apple; Zoe A. Hilbert; Michael S. Kay; Nels C. Elde
Title: Conflicts with diarrheal pathogens trigger rapid evolution of an intestinal signaling axis Document date: 2020_3_30
ID: ju826pao_25
Snippet: 293T cells expressing GC-C variants were grown to confluence in 24-well plates before ligand stimulation. Toxin and uroguanylin solutions were diluted in reduced serum media (Opti-MEM, Thermo Scientific) to the appropriate experimental concentration. For each measurement, cell culture media was then aspirated and replaced with the ligandcontaining solution in 3-6 replicate wells. Following incubation at 37°C/5% CO2 for 20 min, the ligand solutio.....
Document: 293T cells expressing GC-C variants were grown to confluence in 24-well plates before ligand stimulation. Toxin and uroguanylin solutions were diluted in reduced serum media (Opti-MEM, Thermo Scientific) to the appropriate experimental concentration. For each measurement, cell culture media was then aspirated and replaced with the ligandcontaining solution in 3-6 replicate wells. Following incubation at 37°C/5% CO2 for 20 min, the ligand solution was aspirated and cells lysed directly in 0.1 M HCl. Intracellular cGMP was then measured using an Enzyme Linked Immunosorbent Assay kit (Enzo Scientific) according to the manufacturer's specifications. Absorbance was measured using a plate reader (BioTek), and cGMP concentrations calculated based on a standard curve. Four-parameter variable slope dose-response curves were generated in Prism 8 (GraphPad).
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