By Kursad Turksen
ISBN-10: 1493965492
ISBN-13: 9781493965496
Given the range of stories and knowledge that experience instructed the life of heterogeneous populations or subpopulations of stem cells, this unique quantity examines assorted facets of stem telephone heterogeneity. This is going opposed to the long-held guideline that stem cells, outlined via their potential for self-renewal and lineage improvement, comprised a homogenous inhabitants, therefore delivering the reader with a brand new road of exploration into the advanced global of stem phone research. Written for the hugely winning Methods in Molecular Biology series, chapters contain introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and tips about troubleshooting and heading off recognized pitfalls.
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Additional info for Stem Cell Heterogeneity: Methods and Protocols
Example text
4. Centrifuge suspension at 1000 Â g for 5 min. 5. Add MEF freezing medium dropwise (see Note 34). 6. Mix gently. 7. Dispense into cryovials in 1 mL aliquots. 8. Place vials in liquid nitrogen vapor for 1 h (see Note 35). 9. Store vials at À80 C overnight and transfer to a liquid nitrogen storage tank (see Note 36). 10. Thaw one vial for confirmation of cell viability (see Note 37). 4 Sharat Warrier et al. MEF Thawing 1. Incubate fresh T75 tissue culture flasks with 20 mL of MEF culture medium.
If the pellet has changed color to yellow or is too large, resuspend once again in 8 mL of fixative, centrifuge for 7 min at 500 Â g and discard the fixative. If the cell suspension was stored for long, centrifuge for 5 min at 3500 Â g and discard the fixative. Resuspend in fresh fixative until the right turbidity is acquired. 67. The glass slides need to be dust and grease free. Thoroughly clean with detergent and deionized water. 68. A good chromosomal spread should encompass only a few overlapping chromosomes.
Once cooled incubate the slides in SSC solution for 10 min in a water bath at 55 C. 7. Rinse thoroughly three times with deionized water and dry the slides in a 90 C oven for 5 min. 8. While drying the slides, pre-warm 100 mL phosphate buffer in a glass slide holder at 37 C in a warm water bath. Establishment and Characterization of Naı¨ve Pluripotency in Human Embryonic Stem Cells 31 9. Just before use, add 10 μL trypsin stock solution to the phosphate buffer and mix well. Incubate the slides in the trypsin–phosphate buffer solution for 5–8 s at 37 C (see Note 72).
Stem Cell Heterogeneity: Methods and Protocols by Kursad Turksen
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