By William Tse, Kevin D. Bunting (auth.), Kevin D. Bunting (eds.)
With new applied sciences rising and outdated applied sciences being reimagined, the sphere of hematopoietic stem mobilephone (HSC) study keeps to develop swiftly in pursuit of more desirable treatments for plenty of blood illnesses. Updating and development upon the thorough first edition's starting place, Hematopoietic Stem mobile Protocols, moment Edition offers updated protocols built within the HSC box. A group of best researchers offer this quantity with in-depth, with no trouble reproducible tools for potent characterization of HSC and their developmental power. An introductory bankruptcy additionally tricks at strength thoughts which could improve sooner or later. Following the structure of the winning Methods in Molecular Biology™ sequence, every one protocol deals step by step laboratory directions, lists of the mandatory gear, and tips about troubleshooting and keeping off recognized pitfalls.
Essential for the laboratory-based researcher, Hematopoietic Stem mobile Protocols, moment Edition is a far wanted technical source within the seriously vital box of stem cellphone investigation.
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It is up to the individual researcher to learn and develop expertise in the procedures required. 2. 1. Treatment Groups and Controls Animals of the same sex and age should be approximately the same weight. Any significant outliers (alpha dominant mice or runts) should be excluded from experiments. Unless the amount of the factor under evaluation is limiting, 5 mice per group is a good target, although more mice per group improves statistical power as does the performance of replicate experiments.
Minimal essential medium alpha medium ( -MEM, Invitrogen, Carlsbad, CA, USA). 3. 60 mm polystyrene tissue culture dishes. 4. Sterile gloves. 5. Sterile scissors to remove femurs and tibias. 6. Sterile gauze sponges to remove muscle tissues from bones. 7. Sterile 1-ml syringes with 25-G needles to expel bone marrow from medullary cavities. 8. Sterile 5-ml syringe with a 22-G needle to collect flushed marrow. 2. Elutriation 1. Elutriator: we currently use a Beckman J-6M centrifuge with a JE-6B elutriator rotor and a standard chamber.
Another technique that can be used to measure HSPC frequencies in blood involves the use of hematocrit tubes. A standard 75-mm hematocrit tube contains approximately 75 μl of blood when full. Therefore approximately 75 μl of blood containing anticoagulant can be collected into non-heparinized hematocrit tubes, or 75 μl of fresh blood can be collected into heparinized hematocrit tubes and the blood-filled hematocrit tubes centrifuged using a hematocrit microcentrifuge. After centrifugation, the blood is separated into RBC, buffy coat (WBC), and plasma fractions.