By Alexander Meissner PhD, Sarah Eminli PhD, Rudolf Jaenisch PhD (auth.), Julie Audet Ph.D, William L. Stanford Ph.D (eds.)
As regenerative medication contains exchanging diseased cells, tissues or organs, or repairing tissues in vivo, the manipulation of stem cells underlie its targets. In Stem Cells in Regenerative drugs: equipment and Protocols, best specialists within the box offer an up-to-date illustration of the panorama of stem cell-based cures in a large spectrum of tissue structures and ontogenic phases, from the isolation and tradition of stem cells to their genuine use in vivo. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, those chapters contain short introductions to the subject, lists of the required fabrics and reagents, comfortably reproducible, step by step laboratory protocols, and counsel for troubleshooting and keeping off identified pitfalls.
Comprehensive and easy-to-use, Stem Cells in Regenerative drugs: equipment and Protocols is bound to give a contribution vastly to the definition of standardized strategies for the manipulation of somatic and embryonic stem cells in learn and scientific purposes.
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Additional info for Stem Cells in Regenerative Medicine
7) 2. 5 mL Eppendorf Tube. Centrifuge at 3,000 rpm for 3 min. 3. Re-suspend 1Â106 cells in 100 mL of Intraprep Reagent 1 with 50 ml of 2% HF for 15 min at room temperature. 4. Wash 2Â with 1 mL of 2% HF. 5. Dilute primary mouse Oct-4 antibody (1:100) in 100 mL of Intraprep Reagent 2 with 5 mL of 5% BSA. Incubate with cells for 30 min at room temperature. 6. Wash 2Â with 1 mL of 2% HF. 7. Dilute secondary goat anti-mouse IgG-FITC (1:100) in 100 mL of 2% HF with 5 mL of 5% BSA. Incubate with cells for 20 min at room temperature.
Equilibrate two-cell embryos in fusion buffer for 1 min. 15. Transfer embryos between electrodes. 16. Wait until embryos are aligned in AC current ($20 s), AC is ON when the electrodes are connected. 17. Push ‘START’ button. 18. After the two pulses wash embryos through drops of KSOM (pre-equilibrated at 37°C) and return to incubator. 19. Fused cells may appear 5 min after current application, but usually it takes 15–60 min to observe fusion of most embryos. Those that did not fuse must be separated after 1 h and discarded, since they will form regular diploid blastocysts.
4. 5 days post coitum (dpc)). 5. 1% hyaluronidase. Shred oviducts in the drop to release zygotes. 6. Collect cumulus-free zygotes using a mouth pipette. 7. Wash extensively by transferring through several microdrops of M2 medium. 8. After washing, transfer to a new culture dish containing microdrops of pre-equilibrated KSOM under mineral oil and place at 37°C, 5% CO2. 9. 5 dpc (see Note 14). 10. For generating tetraploid embryos (see Fig. 2), zygotes are cultured overnight in KSOM to obtain two-cell embryos.