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 learn have raised the wish that at some point mobile treatment won't in basic terms change lacking neurons, but additionally refill absent chemical indications, metabolites, enzymes, neurotransmitters, or different lacking or faulty elements from the diseased or injured mind. In Neural Stem Cells: tools and Protocols, across the world well-known specialists from educational, medical, and pharmaceutical laboratories describe in nice element the main often used mobile, molecular, and electrophysiological how to isolate, represent, and make the most of neural stem cells. those effortlessly reproducible strategies introduce a number of the assets of stem/progenitor cells, supply quite a lot of stipulations for his or her tradition, and give the chance to outline their houses in tradition. extra thoughts are designed to assist researchers establish endogenous stem cells in addition to exogenous stem cells after transplantation within the mind. The protocols variety from the best tools of isolation and characterization of neural telephone homes to such very refined equipment as characterizing gene expression, telomerase assays, and cellphone cycle kinetics. every one is written by way of an investigator who has used the tactic commonly, and contains step by step directions, tips about averting pitfalls, and helpful notes that make all of the distinction to winning experimental outcomes.
finished and easy-to-follow, Neural Stem Cells: tools and Protocols presents a robust synthesis of trendy key in vitro and in vivo options to spot and represent neural stem cells for examine and for constructing experimental therapeutics.
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Extra info for Neural Stem Cells: Methods and Protocols
Place tube at 4°C for 15 min to harden agar. 6. Cut off the tip of the tube with a razor blade, and use a small spatula to pry the agar plug out. Place the plug into OsO4 for 2 h at room temperature; the osmium will turn the neurospheres brown, and they should then become apparent to the naked eye. 7. Rinse for 30 min at room temperature in H2O, and place into uranyl acetate for 1 h. 8. Dehydrate through graded ethanols, place into embedding plastic, and section for standard transmission EM. See Fig.
Cytosine arabinoside (Ara-C: Sigma) stock of 1 mM in DMEM. 5. 49 +M 3,3v,5-triiodo-L-thyronine (all from Sigma) (13). 6. Anti-GFAP antibody (Dakopatts). Olfactory Bulb Ensheathing Cells 43 Fig. 1. Dissection of cortex for culturing astrocytes. 7. 5 h or overnight in a 37°C incubator. Remove all the PLL and leave the dish/ﬂask to air dry in the hood before use. Dried PLL-coated dishes can be stored for at least 1 wk at room temperature. 3. 1. Primary Culture of Astrocytes for Generating Astrocyte Conditioned Media (ACM) 1.
7. Plate the cells on new ﬁbronectin-coated dishes (see Note 12). 6. Freezing NEP Cells 1. When the dish reaches 80% conﬂuency, remove the culture media. Add 1 mL of trypsin-EDTA solution for 2–3 min at 37°C. 2. Add 3 mL of NEP basal media with 10% CEE to stop trypsinization. 3. Spin the cells down at 300g. 4. Remove the medium. Gently resuspend the cells in 10% DMSO/80% NEP basal medium/10% CEE (4°C) at 106/mL (see Note 13). 5. Transfer the cell suspension to pre-chilled cryogenic vials. Store the vials at –140°C.