By Biju Parekkadan, Martin L. Yarmush
Written and edited by means of well-known specialists within the box, the hot "Artech apartment tools in Bioengineering" ebook sequence bargains certain information on authoritative tools for addressing particular bioengineering demanding situations. providing a hugely functional presentation of every subject, each one publication offers learn engineers, scientists, and scholars with step by step tactics, transparent examples, and powerful how you can conquer difficulties that could be encountered. This state of the art quantity is targeted on the way to derive, manage, objective, and/or organize stem cells for scientific use. The e-book is helping execs grasp robust stem cellphone bioengineering equipment, allowing them to carefully try out hypotheses and examine their effects to 'gold standards'.
The booklet includes step by step tips on how to:
Derive and attempt human embryonic stem cells;
Analyze bone marrow stem cellphone functionality in vitro and in vivo;
Image a stem mobile transplant;
Cryopreserve stem cells;
Differentiate stem cells utilizing microscale strategies.
Read or Download Methods in Bioengineering: Stem Cell Bioengineering (The Artech House Methods in Bioengineering Series) PDF
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Additional info for Methods in Bioengineering: Stem Cell Bioengineering (The Artech House Methods in Bioengineering Series)
Sample text
7. Wash oocytes three times with FHM. 8. Culture oocytes in KSOM+AA for 6 hours in incubator (37°C, 5% CO2, 95% air). 9. Activate oocytes in FHM containing 10 mM calcium ionophore for 5 minutes followed by culturing in KSOM+AA supplemented with 2 mM DMAP for 3 hours in incubator. Wash oocytes three times with FHM. Culture activated oocytes in KSOM+AA in incubator (37°C, 5% CO2, 95% air) for 4 days. Assess blastocyst formation. 2 Generation of p(MII) embryos 1. Give 5 IU (50 mL 100 IU stock) PMSG via intraperitoneal injection into each B6CBA F1 female.
2. 1 µg/mL demecolcine for 1 hour. 3. 56% KCl. 4. Fix cells with 3 methanol:1 acetic acid and then spread onto the slides with Giemsa stain. 2 Immunostaining 1. Add 4% paraformaldehyde to the cells for 15 minutes and then wash with PBS several times. 2. Wash fixed ntES cells in blocking solution consisting of 5% donkey or goat serum (Chemicon) with 5% BSA in PBS for 1 hour at room temperature. 3. Incubate primary antibodies: rabbit polyclonal anti-Oct4 (Santa Cruz Biotechnology), rabbit polyclonal anti-Nanog (Chemicon), rabbit polyclonal Sox2 (Abcam), and mouse monoclonal anti-SSEA-1 (Chemicon) with the cells at room temperature for 1 hour.
When injected subcutaneously into immunodeficient mice, they generate tumors that contain tissues comprising all three germ layers [5]. Mouse parthenogenetic embryos fail to develop beyond the early limb bud stage as a result of the lack of paternal imprints in such embryos [6]. Recently, however, Kono et al. [7] and Kawahara et al. [8] generated live-born pups from mouse parthenogenetic embryos via nuclear transfer and altered expression of imprinted genes. For protocols in which polar body extrusion is blocked, the timing of parthenogenetic activation relative to meiosis will determine the heterozygosity of the resultant pES cell line.