By Kris De Boeck, Melissa Ashlock (auth.), Margarida D. Amaral, Karl Kunzelmann (eds.)
Despite the numerous milestones in cystic fibrosis (CF) learn, growth in the direction of curing the sickness has been sluggish, and it's more and more tricky to know and use the already extensive and nonetheless turning out to be variety of numerous equipment presently hired to check CF with a view to know it in its multidisciplinary nature. Cystic Fibrosis: analysis and Protocols goals to supply the CF examine group and similar researchers with a really wide selection of top quality experimental instruments, as a great way to know and use classical and novel tools utilized to cystic fibrosis. Volume I: techniques to check and proper CFTR Defects makes a speciality of the cystic fibrosis transmembrane conductance regulator (CFTR) and its expression, biogenesis, constitution, and serve as by way of the defects inflicting CF. Written within the hugely winning Methods in Molecular Biology™ sequence structure, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, simply reproducible laboratory protocols, and tips about troubleshooting and keeping off recognized pitfalls.
Comprehensive and functional, Cystic Fibrosis: prognosis and Protocols will offer readers with optimum operating instruments to deal with urgent questions within the top technical method, whereas aiding we all, as a study and medical group, to maneuver speedier hand-in-hand towards unravelling the secrets and techniques of this not easy affliction and medication it.
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Extra info for Cystic Fibrosis: Diagnosis and Protocols, Volume I: Approaches to Study and Correct CFTR Defects
02%) (Euroclone). 6. Transfection reagent: Lipofectamine 2000 (Invitrogen). 7. Synthetic medium to use for DNA–Lipofectamine 2000 complex formation: Opti-MEM (Invitrogen). 2. CFTR and YFP Plasmids Common plasmids or other vectors carrying the CFTR coding sequence and suitable for stable transfections can be used. Plasmids for the YFP-H148Q or YFP-H148Q/I152L can be 16 Pedemonte, Zegarra-Moran, and Galietta obtained from our laboratory or from Dr. S. edu). 3. Saline Solutions 1. 4). 2. 4). 4. Equipment 1.
The selection agents zeocin, hygromycin B (Invitrogen), and geneticin (Calbiochem) are dissolved in tissue culture-grade water at 100 mg/ml, stored in aliquots at −20ºC (zeocin) or at 4ºC (hygromycin B and geneticin), and then added to cells as required. 4. PBS solution without Ca2+ and Mg2+ (Euroclone). 5. 02%) (Euroclone). 6. Transfection reagent: Lipofectamine 2000 (Invitrogen). 7. Synthetic medium to use for DNA–Lipofectamine 2000 complex formation: Opti-MEM (Invitrogen). 2. CFTR and YFP Plasmids Common plasmids or other vectors carrying the CFTR coding sequence and suitable for stable transfections can be used.
Forskolin, CPT-cAMP, and potentiators). 2. Microplates are transferred to a microplate reader for CFTR activity determination. 3. The assay in each well consists of a continuous 14 s fluorescence reading with 2 s before and 12 s after injection of 165 μl of the iodide-rich Dulbecco’s PBS. This step ensures that the cells are exposed to a final iodide concentration of 100 mM. Injection flow rate is set at 100–160 μl/s. Cell incubation and assay are carried out at 37◦ C. 4. CFTR Assay by Fluorescence Microscope This method has been successfully used to study the effect of CFTR inhibitors (10) and potentiators (11) after transient transfection in cells like HEK-293: 1.
Cystic Fibrosis: Diagnosis and Protocols, Volume I: Approaches to Study and Correct CFTR Defects by Kris De Boeck, Melissa Ashlock (auth.), Margarida D. Amaral, Karl Kunzelmann (eds.)