By Kursad Turksen
Now in volumes, this thoroughly up-to-date and elevated version of Embryonic Stem Cells: equipment and Protocols presents a various number of quite simply reproducible mobile and molecular protocols for the manipulation of nonhuman embryonic stem cells. quantity , Embryonic Stem phone Protocols: Differentiation types, moment variation, covers cutting-edge equipment for deriving many varieties of differentiating cells from ES cells. the 1st quantity, Embryonic Stem mobile Protocols: Isolation and Characterization, moment variation, offers a various choice of with ease reproducible mobile and molecular protocols for the isolation, upkeep, and characterization of embryonic stem cells. Together, the 2 volumes remove darkness from for either rookies and specialists our present knowing of the biology of embryonic stem cells and their software in basic tissue homeostasis and regenerative medication purposes.
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Extra resources for Embryonic Stem Cell Protocols, Vol. II: Differentiation Models, 2nd Edition, v330
Natl. Acad. Sci. USA 91, 4303–4307. , and Monk, M. (1987) HPRT-deficient 50. (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature 326, 292–295. , and Bartlett, P. F. (1990) Fibroblast growth factor stimulates the 51. proliferation and differentiation of neural precursor cells in vitro. J. Neurosci. Res. 25, 463–475. 52 Wagner, J. , Black, I. , and DiCicco-Bloom, E. (1999) Stimulation of neonatal and adult 52. brain neurogenesis by subcutaneous injection of basic fibroblast growth factor.
Microscope slides. 14. Tissues. 15. Nail polish. 16. Microscopes. Cover slips were examined with a Zeiss Axioplan 2 fluorescence microscope. Nunclon multidishes were examined with a Leica DMIRB inverted fluorescence microscope. 3. Methods To obtain a homogeneous population of neural precursor cells from which oligodendrocyte lineage cells originate, we used genetically engineered ES cells that allow the selection of such committed cells. Specifically, we used doubly targeted ES cells so that, after induction of differentiation, we could select negatively against residual ES cells and select positively for neural precursor cells.
2 mL) and keep at –20°C. 17. OPC differentiation medium (Sato base): combine 20 mL DMEM, 200 µL PSG, 200 µL 100X Sato stock solution, 200 µL 100X N-acetyl-cysteine, 4 µL 5000X forskolin, 20 µL 1000X biotin, 20 µL 1000X insulin, 20 µL 1000X NT3. Keep this medium for 1–2 wk at 4°C. Add growth factor and drugs when needed. 4. OLIGODENDROCYTE DIFFERENTIATION 1. 1000X PDGF-AA (10 µg/mL): dilute 2 µg human PDGF (Peprotech, cat. no. 2 mL filter-sterilized 10 mM acetic acid. Aliquot (20 µL) and store at –80°C.