By Ivan N. Rich (eds.)
This volumepresents updated equipment that permit basic stem cells from a number of assets to be remoted, cultured in vitro, detected and measured for particular functions. those functions diversity from these in simple, stem phone and veterinary learn to toxicology, mobile remedy and regenerative medication. there's a mild bias in the direction of the blood-forming process as extra is understood concerning the blood-forming or hematopoietic method than the other fundamental stem cellphone procedure. those certain houses and features are mentioned and tested, as a rule on the mobile point and intimately during this booklet. Written within the winning Methods in Molecular Biology sequence structure, chapters contain introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, conveniently reproducible protocols, and notes on troubleshooting and fending off recognized pitfalls.
Authoritative and available, Stem cellphone Protocols offers newbies with the basics essential to enhance new applied sciences worthy for simple and scientific examine sooner or later, and may reduction pros to find new methodologies to supply a much wider standpoint and an excellent larger scope for his or her personal research.
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Extra resources for Stem Cell Protocols
3. Alexa Fluor® 700-conjugated anti-mouse c-Kit (CD117) antibody. 4. Pacific blue (PB)-conjugated anti-mouse Sca-1 (Ly6A/E) antibody. 5. Phycoerythrin (PE)-conjugated (CD135) antibody. anti-mouse Flk2/Flt3 6. Fluorescein-5-isothiocyanate (FITC)-conjugated anti-mouse CD34 antibody. 7. Alexa Fluor® 647 anti-mouse CD150 (SLAM) antibody. 8. Hoechst 33342 is a DNA dye that can be pumped out by stem and progenitor cells. Stem/progenitor cells thus have a 24 Yi Liu et al. Hoechst low fluorescence in both the blue and red regions of the spectrum.
4 Stem Cell Marker Staining 27 1. Add CD34-FITC to lineage-depleted cells (main sample) at a concentration of 1:25. Incubate for 90 min at 4 °C on a rocker. Add 2 μL of CD34-FITC into Control 3 tube. 2. Incubate for 1 h, and add other markers for stem cell staining. The panel of markers and the corresponding concentrations used in the authors’ laboratory are Sca-1 (1:100), c-Kit (1:100), Flk2/ Flt3 (1:100), and CD150 (1:100). Streptavidin is also added to the cell suspension to bind biotinylated lineage antibodies (1:150).
1 mL using non-sterile tips. 8. Electronic cell counter or flow cytometer that can determine nucleated cell counts. 9. , hemocytometer, flow cytometer, viability counter). 10. Sterile, conical 15 and/or 50 mL tubes with screw lid. 11. Sterile, 5 mL tubes with snap on lid. 12. 5 mL Eppendorf conical vials. 2 Reagents 37 1. Dulbecco’s phosphate buffered saline (dPBS). 2. Iscove’s Modified Dulbecco’s Medium (IMDM). 3. 077, Axis-Shield; or Ficoll, GE Healthcare). 4. Cryopreservation medium: IMDM + 10 % fetal bovine serum (FBS) + dimethylsulfoxide (DMSO).