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Tham khảo tài liệu 'atomic force microscopy in cell biology episode 1 part 9', kỹ thuật - công nghệ, cơ khí - chế tạo máy phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | 164 Davis et al. 10. Perfusion apparatus standard system for retrograde coronary perfusion see ref. 24 for details . 11. DMEM solution Life Technologies UK . 12. Serum substitute Ultroser G Life Technologies UK cat. no. 81-003 . 13. Collagenase Worthington Type CLS I Lorne Laboratories UK . 14. Glutaraldehyde Sigma-Aldrich . 15. 10-mL Sterile tubes Sigma-Aldrich cat. no. C-3084 . Centrifuge Note with heart cells small benchtop centrifuges are not to be recommended because their rapid acceleration can damage myocytes. Much better are the larger free-standing cooled types such as the Mistral 4L. 3. Methods The methods described below outline the process of 1 cell preparation isolation and electron microscopy characterization and 2 scanning force characterization. 3.1. Cell Preparation and Electron Microscopy Characterization The detailed tissue dissociation protocols for the adult heart 24 will provide isolated myocytes suspended in Dulbecco s modified Eagle s medium supplemented with pyruvate and 10 mM HEPES to which has been added 2 v v serum substitute. It is convenient to store cells at room temperature in sterile 10-mL tubes from Sigma-Aldrich. As long as standard precautions have been taken during isolation these myocytes can be used for up to 24 h without any antibiotics. When incubation at 37 C is used 100 O2 with HEPES media or 95 O2 5 CO2 v v if bicarbonate buffered only then antibiotics must be added. Myocytes can be plated directly on to cover slips. Alternatively cells can be fixed by first resuspending in Krebs buffer NaCl 134.1 KCl 5.4 NaH2PO4.2H2O 0.3 MgCl2 1 Na acetate.3H2O 5 Na pyruvate 5 glucose 11.1 HEPES 5 all mM pH adjusted to 7.4 with NaOH and then adding 1 volume of myocytes to 2.5 v v glutaraldehyde in 1 3 Krebs buffer. Though fixation is rapid the cells are typically left for at least 30 min then resuspended in phosphate-buffered saline and stored at 4 C. This fixative procedure has been designed to reduce the osmotic strength of the .
