By Lichao Luo, Phing Chian Chai, Yu Cai (auth.), Kursad Turksen (eds.)
Interest in a really expert microenvironment or “niche” regulating hemopoietic stem mobilephone functionality has been progressively becoming because the thought used to be first proposed through Ray Schofield over 3 many years in the past. This becoming curiosity, in addition to extra lately the curiosity in cellular-molecular-biochemical characterization of not just the hemopoietic stem mobile area of interest however the niches for different stem cells, incited the compilation of Stem telephone area of interest: tools and Protocols. during this quantity, scientists have supplied protocols that would supply either a style of the sector and confidently stimulate new techniques and methodologies via these drawn to the stem mobile area of interest. Written within the profitable Methods in Molecular Biology sequence structure, chapters contain introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, with no trouble reproducible protocols, and notes on troubleshooting and keeping off identified pitfalls.
Authoritative and simply available, Stem mobile area of interest: tools and Protocols seeks to serve either specialists and beginners within the stem mobilephone box with well-established protocols in this intriguing topic.
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Extra resources for Stem Cell Niche: Methods and Protocols
Sample text
1:1 mix of IMDM and αMEM supplemented with 10 % FCS, 1 % P/S, and 1 % L-glutamine. 5 Preparation of 3D Coculture System 1. Positive displacement pipette. 2. 5 and 15 mL sterile centrifuge tubes. 3. 24-well plates. 46 Brahmananda Reddy Chitteti et al. 4. Type I collagen oligomers acid-extracted and purified from the dermis of porcine skin [19, 20]. Collagen concentration is determined using a Sirius Red (Direct Red 80) assay as previously described [21]. Collagen formulations are standardized based upon purity as well as polymerization potential [20].
1 Analysis Laser scanning confocal microscope is used for analysis. 1 Adult Ovaries All steps are carried out at room temperature unless otherwise stated. During all incubations and washes, the Eppendorf tubes are placed on a nutator. 1. Immobilize 5–10 female flies by putting them on an ice block. 2. The ovaries are positioned in the abdomen of the fly and are simple to find in well-fed individuals (see Note 12). Dissect the flies in 1× PBS using a stereomicroscope, and hold the fly with one pair of tweezers at the thorax.
Place a slide under dissecting microscope and put drops of dissecting solution on the slide. 3. Take the males and females using tweezers. Put one tweezers at the thorax region and with other tweezers take out the terminalia and isolate the ovaries and testes. 4. Transfer the dissected testis and ovary into separate tubes containing dissecting solution in ice. 5. For ovary, it is better to dissociate ovaries into ovarioles and then fragment each ovariole into pieces with fine tungsten needles before putting in dissecting solution or fixation solution.