By Mark T. Clunes, Richard C. Boucher (auth.), Margarida D. Amaral, Karl Kunzelmann (eds.)
Despite the numerous milestones in cystic fibrosis (CF) learn, development towards curing the disorder has been gradual, and it really is more and more tough to understand and use the already extensive and nonetheless transforming into diversity of various tools at present hired to review CF so one can comprehend it in its multidisciplinary nature. Cystic Fibrosis: analysis and Protocols goals to supply the CF examine group and comparable researchers with a truly wide variety of high quality experimental instruments, as an ideal way to know and use classical and novel tools utilized to cystic fibrosis. Volume II: tools and assets to appreciate Cystic Fibrosis specializes in pathophysiology, Omics methods, and quite a few key assets lately made on hand for CF learn. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, easily reproducible laboratory protocols, and pointers on troubleshooting and heading off identified pitfalls.
Comprehensive and useful, Cystic Fibrosis: analysis and Protocols will offer readers with optimum operating instruments to deal with urgent questions within the most sensible technical manner, whereas supporting we all, as a study and medical group, to maneuver swifter hand-in-hand towards unravelling the secrets and techniques of this difficult affliction and remedy it.
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Additional resources for Cystic Fibrosis: Diagnosis and Protocols, Volume II: Methods and Resources to Understand Cystic Fibrosis
Example text
When ENaC and CFTR are coexpressed, amiloride-sensitive currents can be measured before activation of CFTR and then repeated after activation of CFTR. The effect of these reagents on water-injected oocytes should be used as a control for endogenous currents. This can allow the isolation of CFTR or ENaC currents in the absence or the presence of the other. 5. In oocytes expressing CFTR alone, currents are not sensitive to 10 μM amiloride before or after activation of CFTR. Activation of CFTR by forskolin and IBMX leads to an increased outward chloride flux.
2. Culture of Cell Lines Heterologously Expressing CFTR BHK-21 cells stably expressing CFTR, F508del, and GFP- or extracellularly HA epitope-tagged variants [Extope-CFTR; (6)] are grown in DMEM/F12, 5% FBS, and 500 μM methotrexate at low confluence on glass bottom microwell dishes (MatTek Corporation, USA) or BD BioCoat collagen-coated culture slides (BD Biosciences, USA) (see Note 1). In cell lines stably expressing CFTR, the levels of CFTR protein expression can be increased CFTR Localization 21 by addition of 2 mM sodium butyrate to the growth medium for 8–16 h before the experiment.
Connect the trans chamber of the bilayer system to the current–voltage converter using an Ag–AgCl electrode and 3 M KCl–3% agar. 6. Connect the cis chamber of the bilayer system to the voltage source using an Ag–AgCl electrode and 3 M KCl–3% agar. 7. Use “painting” method to form bilayer over the septum aperture using the lipid containing membrane-forming solution in n-decane described in Step 3 (see Note 12). 8. Establish the bilayer membrane formation by following membrane capacitance. 9. Place a small aliquot of the oocyte vesicle suspension into the trans chamber and wait for fusion to occur (see Note 13).