By Milica Radisic, Lauren D. Black III
Cardiac Tissue Engineering: equipment and Protocols presents a set of protocols on cardiac tissue engineering from pioneering and best researchers around the world. those contain tools and protocols for mobile instruction, biomaterial instruction, telephone seeding, and cultivation in numerous structures. Written within the hugely winning Methods in Molecular Biology sequence layout, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, easily reproducible laboratory protocols, and key tips about troubleshooting and warding off identified pitfalls.
Authoritative and functional, Cardiac Tissue Engineering: tools and Protocols highlights the foremost concepts, either experimental and computational, for the learn of cardiovascular tissue engineering.
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Extra info for Cardiac Tissue Engineering: Methods and Protocols
2. 1 % BSA–PBS in a 5 mL round-bottom Falcon tube. 42 Kristin M. French and Michael E. Davis 3. Concentrate the beads on the magnetic sorter. 4. 1, steps 3 and 4, is one wash) (see Note 4). 5. Add 5 μL of c-kit antibody (see Note 2). 6. Incubate on a rotator for 2 h at 37 °C. 7. 1 % BSA–PBS. 8. Resuspend the beads in 2 mL of same solution and store at 4 °C. 9. Use the antibody-conjugated beads within 2 weeks. 2 Isolation All animal work should be approved by the appropriate institutional committee.
The isolation, expansion, and characterization of CPCs are summarized in Fig. 1. 6). 8) samples. 1 Materials MACS 1. Tissue. 2. Sterile surgical equipment. 3. Dyna beads (Dynal) (see Notes 1 and 2). 4. Magnetic sorter (Dynal). 5. 5 mL Falcon tubes, round bottom, with lids. 6. C-kit H-300 (Santa Cruz), sterile. 7. Hanks’ balanced salt solution (HBSS), sterile. 8. 1 % Bovine serum albumin (BSA)–phosphate buffered saline (PBS), sterile. 9. Collagenase: 1 mg/mL in HBSS, pH 7; sterile. 10. 70 μm Cell strainer.
Culture until the clonal candidates reach 80 % confluency in the T75 culture flasks, 1–2 weeks. Exchange media every 7 days. 27. The cells are now ready for characterization and further expansion of c-kit-positive clones. 28. Trypsinize and pellet the cells. 29. Resuspend the cells in culture media and count. 30. Set aside 1 × 105 cells per desired stain (at least 5 × 105 cells) for characterization. 31. Seed the remaining cells at 5 × 105 to 1 × 106 cells per T75 culture flask (see Note 13). 32.