By Teresa V. Bowman, Akil A. Merchant (auth.), Meral Beksac (eds.)
ISBN-10: 1588295958
ISBN-13: 9781588295958
ISBN-10: 1597452238
ISBN-13: 9781597452236
Molecular equipment: Stem phone Transplantation provides a compendium of state-of-the-art learn at the molecular steps focused on hematopoietic stem cellphone (HSC) activation and self-renewal. The emergence of HLA typing, and the optimistic influence it has had at the luck of scientific transplantation is emphasised and mentioned through impressive stem cellphone researchers.
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Extra resources for Bone Marrow and Stem Cell Transplantation
Sample text
Gently rock the culture dish to mix and return the cells to the incubator overnight. DAY 3: 8. Check the cells under a microscope. Small black particles should be visible. 9. Remove the transfection media. Replace with 10 mL fresh growth media. 30 Duda et al. DAY 5: 10. 45-µm pores. 11. Centrifuge the supernatant at 22,000g for 2 h to concentrate the virus (see Note 8). 12. Resuspend the viral pellet in 250 µL/10-cm plate of packaging cells. Use myelocult media. 13. Test the concentrated virus for optimal dilution by adding it to the supernatant of 293T cells at dilutions of 1:1, 1:10, and 1:100.
Mix gently and incubate for 20 min to 6 h at RT. 4. Add the prepared 1 mL of DNA-Lipofectamine 2000 complexes to each 6-cm plate containing cells and 5 mL of medium. Mix gently by rocking back and forth. 5. Incubate the cells at 37°C/5% CO2 for 48 h before harvesting viral supernatant. DAY 5: 6. Euthanize donor mice according to approved animal protocols. 7. Harvest bilateral femurs and tibias from each mouse. Scrape bones with a razor blade to remove any attached muscle, cartilage, and connective tissue.
Add 11 µg lentiviral expression plasmid, 3 µg lentiviral packaging plasmid, and 6 µg of lentiviral envelope plasmid. Add deionized H2O to a final volume of 500 µL. 5. Slowly add the DNA/calcium mixture drop–wise to the 2X HBS solution while gently vortexing the 15-mL tube. The mixture must not spill. 6. Gently vortex for 10–15 min to allow the formation of calcium phosphate/DNA precipitates. 7. Remove the packaging cells from incubator at the last moment. Add the mixture one drop at a time to the supernatant.
Bone Marrow and Stem Cell Transplantation by Teresa V. Bowman, Akil A. Merchant (auth.), Meral Beksac (eds.)
by Michael
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