Animal cell culture techniques pdf
Cell isolation and culture are essential tools for the study of cell function. Isolated cells grown under controlled conditions can be manipulated and imaged at a level of animal cell culture techniques pdf that is not possible in whole animals or even tissue explants. Recent advances have allowed for large-scale isolation and culture of primary C. Isolated cells can be used for single-cell profiling, electrophysiology, and high-resolution microscopy to assay cell autonomous development and behavior.
This chapter describes protocols for the isolation and culture of C. Our protocols describe isolation of embryonic and L1 stage cells from nematodes grown on high-density NA22 bacterial plates and isolation of L2 through L4 stage cells from nematodes grown in axenic liquid culture. Both embryonic and larval cells can be isolated from nematode populations within 3 hours and can be cultured for several days. A primer on sterile cell culture techniques is given in the appendices.
The methodology of culturing isolated cells has been available for over a century. By the 1950s, the introduction of protease digestion, defined media, and antibiotics made vertebrate cell culture a more widely accessible technique. The main difficulty has been that the chitinous shell of embryos and the tough outer cuticle of larval and adult worms are resistant to cell and tissue explantation. In contrast to embryos, the tough cuticle of larval and adults nematodes formed an almost impenetrable barrier to post-embryonic cell culture. The protocols collected here provide comprehensive techniques and media recipes for large-scale isolation and culture of primary embryonic and larval cells from C. We suggest equipment and supplies for cell culture in Appendix A and Appendix B.
We include common cell culture techniques in Appendix C for those unfamiliar with working in a sterile culture hood. Both embryonic and larval cell isolations require 30 to 50 µl of compacted eggs or larvae per preparation. NA22 bacteria grow in thick layers on peptone-augmented agar plates and provide a richer food source than do OP50 bacteria, which is standardly used for nematode maintenance. 1 M potassium phosphate pH 6. 8 L ddH2O, and adjust pH to 6. Bring to 1 L in ddH2O and autoclave. 2X YT media may also be used to grow NA22 bacteria.