By K. Sue O’Shea (auth.), Tanja Zigova PhD, Paul R. Sanberg PhD, DSc, Juan R. Sanchez-Ramos PhD, MD (eds.)
During the last decade, advances in neural stem mobilephone study have raised the desire that in the future mobile remedy won't purely exchange lacking neurons, but in addition fill up absent chemical indications, metabolites, enzymes, neurotransmitters, or different lacking or faulty elements from the diseased or injured mind. In Neural Stem Cells: equipment and Protocols, the world over famous specialists from educational, medical, and pharmaceutical laboratories describe in nice aspect the main often used mobile, molecular, and electrophysiological how to isolate, signify, and make the most of neural stem cells. those with no trouble reproducible ideas introduce a few of the assets of stem/progenitor cells, offer quite a lot of stipulations for his or her tradition, and give the chance to outline their houses in tradition. extra suggestions are designed to aid researchers establish endogenous stem cells in addition to exogenous stem cells after transplantation within the mind. The protocols diversity from the easiest equipment of isolation and characterization of neural mobile houses to such very refined equipment as characterizing gene expression, telomerase assays, and telephone cycle kinetics. each one is written by way of an investigator who has used the tactic broadly, and contains step by step directions, tips about warding off pitfalls, and useful notes that make all of the distinction to winning experimental outcomes.
entire and easy-to-follow, Neural Stem Cells: equipment and Protocols offers a strong synthesis of ultra-modern key in vitro and in vivo thoughts to spot and symbolize neural stem cells for examine and for constructing experimental therapeutics.
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14. Suslov, O. , Kukekov, V. , Laywell, E. , and Steindler, D. A. (2000) RT-PCR amplification of mRNA from single brain neurospheres. J. Neurosci. Meth. 96, 57–61. 15. , Lim, D. , Garcia-Verdugo, J. , and Alvarez-Buylla, A. (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97, 703–716. Stem Cells, Neuroblasts, and Glioblasts from Fetal Tissue 29 3 Isolation of Stem and Precursor Cells from Fetal Tissue Yuan Y. Wu, Tahmina Mujtaba and Mahendra S. Rao 1.
No. D-2650). 3. Antibodies 1. Anti-E-NCAM antibody, obtained from 5A5 hybridoma (Developmental Studies Hybridoma Bank) supernatant, 1Ϻ1 in NEP basal medium. 32 Wu, Mujtaba, and Rao 2. Anti-A2B5 antibody, obtained from A2B5 hybridoma (ATCC, cat. no. CRL-1520) supernatant, 1Ϻ1 in NEP basal medium. 3. Anti-nestin antibody, obtained from rat-401 hybridoma (Developmental Studies Hybridoma Bank) supernatant, 1Ϻ1 in NEP basal medium. 4. Anti-`-III-tubulin, 1Ϻ500 (Sigma, cat. no. T8660). 5. Goat anti-mouse UNLB-IgM (Southern Biotechnology, cat.
Dissociate the cells by gently triturating. 8. Plate the cells at 50% confluence on poly-L-lysine/laminin- or fibronectin/ laminin-coated dish in NEP basal medium. 3. Maintenance and Testing of Lineage-Restricted Precursor Cells 1. 2. 3. 4. Incubate the spinal cord cells at 37°C in a 5% CO2. To keep the cells dividing, add 20 ng/mL of FGF every 2 d. To enhance neuronal cell survival, add 10 ng/mL of NT-3 every 2 d. Test the immunocytochemical properties of the cells by staining the cells with anti-A2B5, -NCAM, and -`-III-tubulin antibodies.