By Andrei I. Ivanov
This quantity presents a entire number of classical and leading edge protocols and strategies to check the traditional improvement and physiological capabilities of the gastrointestinal approach and to version the commonest digestive diseases. The chapters concentrate on different examine issues together with ex vivo platforms to check gastrointestinal improvement and capabilities, in vivo imaging of the gastrointestinal tract, isolation and characterization of intestinal immune cells, and animal versions of gastrointestinal irritation and melanoma. The Gastrointestinal body structure and illnesses: tools and Protocols book targets broad viewers of physiologists, mobilephone and developmental biologists, immunologists, and physician-scientists operating within the box of gastroenterology and beyond. Written within the hugely successful Methods in Molecular Biology series layout, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, quite simply reproducible laboratory protocols, and pointers on troubleshooting and fending off identified pitfalls.
Highly useful and obviously written, Gastrointestinal body structure and ailments: equipment and Protocols will serve either professional researchers in addition to novices to the sphere and may offer a special source and professional counsel to fashionable laboratory ideas built for interpreting general services and ailments of the gastrointestinal tract.
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Extra info for Gastrointestinal Physiology and Diseases: Methods and Protocols
Sample text
Set the tube back on ice briefly to let larger fragments of tissue settle to the bottom of the tube, but not long enough for gastric glands to settle. Using a 1000 μL pipet, remove the 5 mL of volume without disturbing the large tissue fragments at the bottom of the tube. Transfer this volume into a sterile 5 mL tube and centrifuge at 150 g for 5 min at 4 °C. 6. Remove supernatant carefully, so as not to disturb the pellet of glands (see Note 6). Keeping the tube on ice, transfer desired volume of Matrigel™ to the glands (see Note 7) and mix gently to avoid air bubbles.
Remove supernatant carefully, so as not to disturb the pellet of glands (see Note 6). Keeping the tube on ice, transfer desired volume of Matrigel™ to the glands (see Note 7) and mix gently to avoid air bubbles. 7. Using a 200 μL pipet, pipet 40 μL of the glands in Matrigel™ directly onto the Transwell™ membrane (see Note 8). Incubate the plate at 37 °C for 15 min to allow Matrigel™ to solidify. 5 mL of growth media to the top of the transwell, and 1 mL of growth media to the bottom of the transwell.
2. 1× Organoid culture media: Advanced DMEM/F12 supplemented with Penicillin, Streptomycin, Fungizone, L-glutamine, 50 ng/ml EGF (Peprotech, #315-09), 250 ng/ml R-Spondin1 (R&D Systems, #3474-RS), 100 ng/ml Noggin (Peprotech, #250-38), 1 μM Jagged-1 (ANASPEC, #61298), 10 μM Y27632. Store at 4 °C. 3. 12-well culture plate. 4. Accumax™ (Innova Cell Technologies). Store at 4 °C (see Note 2). 5. 4. Store at 4 °C. Lentiviral Infection of Organoids 15 6. 2× Organoid culture medium: This medium has the same composition as the 1× Organoid medium, but contains twofold higher concentrations of all growth factors and inhibitors.