This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.
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Method Article
Homing of Hematopoietic Cells to the Bone Marrow
In This Article
Summary
Abstract
Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.
Protocol
- We begin the homing experiment by extracting cells to be used in the experiment. For this current experiment we are interested in finding out if the homing of whole bone marrow to the niches in the long bones of recipient animals is different for bone marrow cells extracted from WT animals such as C57BL/6J (WT) and those that are transgenic knockouts for Lysophosphatidic Receptor 1 (LPAR1) (KO).
- Before we start the experiment, we will collect the materials we need for this experiment. In our lab all mouse dissections are carried out in a laminar flow tissue culture hood especially if the cells are going to be transplanted into a recipient animal instead of being used for....
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Discussion
Homing is the process whereby bone marrow cells including stem cells, progenitor cells and differentiated cells, their way into the bone marrow after being injected into the blood stream or bone marrow cavity of mice. As is obvious from the definition, a scientist may chose to study the homing of hematopoietic stem cells, progenitor cells or differentiated cells identified by immunophenotyping and isolated by cell sorting. Typically scientists inject bone marrow cells depleted for lineage markers and hence enriched for.......
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Acknowledgements
This protocol was tested and optimized in the laboratory of Dr. David Scadden. Funding is provided by the Dana Farber Cancer Institute/Massachusetts General Hospital Clinical Hematology and Oncology Fellowship Program.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| DPBS (Dulbecco’s Phosphate Buffered Saline | Mediatech, Inc. | 21-031-CV | |
| Sterile Fetal Bovine Serum | Valley Biomedical, Inc. | BS3033 | |
| 0.5M EDTA (pH 8.0) | Boston BioProducts | BM-150 | |
| Tryphan Blue solution | Mediatech, Inc. | 25-900-CI | |
| ACK Lysing Buffer | Lonza Inc. | 10-548E | |
| Isopropranolol U.S.P. | Denison Pharmaceuticals, Inc | ||
| Vybrant DiD cell-labelling solution | Invitrogen | V22887 | |
| Vybrant DiI cell-labelling solution | Invitrogen | V22885 |
References
- Adams, G. B., et al. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature. 439 (7076), 599-603 (2006).
- Broxmeyer, H. E. Chemokines in hematopoiesis. Curr Opin Hematol. 15 (1), 49-58 (2008).
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