Authors
Istvan Blazsek, Denis Clay, Philippe Leclerc, Jozsef Blazsek, Jean-Jacques Candelier, Csaba Dobo-Nagy, Ibrahim Khazaal, Bruno Péault, Georges Uzan, and Marie-Caroline Le Bousse-Kerdilès
Summary
Self-renewing organs in adult mammals are composed of numerous tissue-specific functional units, such as intestinal crypts/villi or hair follicles, which all involve stem cell (SC) niches. Analogous tissue units in haematopoietic systems, however, have remained elusive. We design here a step-by-step protocol for in situ mapping, purification, enumeration and structural-functional analysis of the haematopoietic tissue unit, termed haematon. Longitudinal bisection of mouse femur and careful, limited dispersion of bone marrow (BM) parenchyma reveals compact, node-like haematons at discreet capillary loops along the diaphysis and spongy metaphysis. Recovery and fractionation of the whole BM in a bulk cell suspension, haematon units and endosteal layer provides a reproducible tool for quantitative and topographical analysis of putative SC niches in defined tissue compartments. We show examples how to characterize haematons in native state or following long-term culture using laser-scanning confocal microscopy, flow cytometry, clonal bioassays and videomicroscopy.Further details
The protocol was published on Protocol Exchange on 10 January 2013. To see the entire protocol, click on the source link.Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 321
- Comments 0