By Kris De Boeck, Melissa Ashlock (auth.), Margarida D. Amaral, Karl Kunzelmann (eds.)
Despite the numerous milestones in cystic fibrosis (CF) study, growth in the direction of curing the disorder has been gradual, and it's more and more tricky to know and use the already broad and nonetheless becoming diversity of various tools at present hired to check CF with a view to are aware of it in its multidisciplinary nature. Cystic Fibrosis: prognosis and Protocols goals to supply the CF study group and similar researchers with a really wide selection of fine quality experimental instruments, as an effective way to know and use classical and novel tools utilized to cystic fibrosis. Volume I: methods to check and proper CFTR Defects specializes in 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 layout, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and tips about troubleshooting and keeping off recognized pitfalls.
Comprehensive and useful, Cystic Fibrosis: prognosis and Protocols will offer readers with optimum operating instruments to handle urgent questions within the top technical method, whereas supporting we all, as a learn and scientific group, to maneuver quicker hand-in-hand towards unravelling the secrets and techniques of this tough ailment and healing it.
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Additional info for Cystic Fibrosis: Diagnosis and Protocols, Volume I: Approaches to Study and Correct CFTR Defects
2000) Mechanism and cellular applications of a green fluorescent protein-based halide sensor. J. Biol. Chem. 275, 6047–6050. 3. Galietta, L. , Haggie, P. , and Verkman, A. S. (2001) Green fluorescent protein-based halide indicators with improved chloride and iodide affinities. FEBS Lett. 499, 220–224. 4. , Thiagarajah, J. , Sonawane, N. , Galietta, L. , et al. (2002) Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J.
After treatment, cells were acutely stimulated with forskolin (20 μM) and genistein (50 μM). In both panels, the arrow indicates iodide addition. 2. 1. Cell Culture Media and Transfection Reagents 1. Cell culture medium for FRT cells: Coon’s modification of F-12 (Sigma-Aldrich). This is a powder medium that requires addition of sodium bicarbonate as indicated by the supplier. After solubilization, the medium is supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum.
To prepare membranes, wash the cells three times with 6 ml of PBS. All steps are performed on ice or at 4◦ C. We use 13-ml polypropylene tubes and centrifuge at 3,000×g for 3 min each time. Suspend the cells in 3 ml of LIS buffer. Transfer into a 7-ml Dounce homogenizer and break cells using 40 strokes with a glass “loose” homogenizer. Add 3 ml of solution A followed by 15 additional strokes. Transfer the broken cells back to the 13-ml tube and centrifuge at 6,000×g for 5 min. Transfer the supernatant to a 30-ml polycarbonate tube containing 20 ml of TBS.
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.)