Fig. 1
Illustrative example of bone marrow transplantation to generate mixed chimeras in mice. Six- to eight-week-old recipient mice are irradiated with myeloablative doses of radiation. In this example, femurs from non-fluorescent and transgenic fluorescent DsRed donors are extracted following euthanasia. One day after irradiation, BM cells are processed and mixed in equal numbers and are injected intravenously in recipient mice. Six weeks following BMT, chimerism in surviving recipients is analyzed. Irradiated mice exhibit visual signs such as the appearance of grey patches of hair on skin. Reconstitution can be confirmed by analyzing the blood counts of the recipient mice using a hematological counter. In this example, the engrafted donor cells can be distinguished as derived from the DsRed transgenic- or the wild-type donor mice based on their fluorescence in the DsRed channel by flow cytometry or by intravital microscopy. Flow cytometry allows for standardized quantification and characterization of relative numbers of the different donor-derived cells in the recipients. Intravital microscopy allows the direct visualization of their relative numbers and to detect possible differential behaviors of distinct donor-derived hematopoietic cell subtypes. Both protocols could therefore be used to visualize not only the efficiency of engraftment of irradiated recipients but also for the analysis of functional differences between the donor cell subsets during atherosclerosis
2 Materials
1.
Irradiator: A commercial irradiator of X-rays or gamma rays with a cesium/cobalt source.
2.
Atherosusceptible recipient mice (e.g., Apoe−/−, or Ldlr−/−).
3.
Sterile phosphate-buffered saline (PBS).
4.
Saline solution or cell culture medium (e.g., RPMI) without antibiotics.
5.
Scalpel.
6.
Petri dishes.
7.
Hemocytometer.
8.
0.5–1 ml syringes.
9.
21 and 25–27 G needles.
10.
Automatic pipettes for resuspension.
11.
General anesthetic (e.g., isoflurane 2 %).
12.
Flow cytometer: To check bone marrow reconstitution by staining blood cells with antibodies for specific markers (e.g., CD45.1).
13.
Hematological cell counter: For analysis of blood parameters.
14.
Optional: Microscope and surgical tools if intravital microscopy needs to be performed.
3 Methods
3.1 Preparation of Donor BM Cells
1.
Sacrifice donor mice and obtain femurs (and tibiae if necessary). For reference, up to 30 × 106 cells can be obtained from one femur of a healthy donor. BM cellularity increases with age, but declines in very old mice. Hence the age of the donor mice should be preferably between 6 and 14 weeks (see Note 1 ).
2.
Use a scalpel to make an incision at the head of the bones.
3.
Flush bone marrow in a laminar flow hood, using PBS or cell culture medium (containing no serum). Use a 21 G needle for femurs, and 25–27 G for tibiae (see Note 2 ).
4.
Homogenize marrow gently with a p1000 pipette or a 1-mL syringe with 21 G needle, taking care not to break cells. Make a single-cell suspension. Lysis of red blood cells using hypotonic buffers is possible but not recommended in order to preserve the full viability of BM cells.
5.
Count cells with the hemacytometer and adjust with PBS or sterile saline solution to have 1 × 106 BM cells in 100 μL (the volume to be injected in the recipient mice). This number should be adjusted depending on existing information about the quality of hematopoietic stem cells in the donor mice (see Notes 3 and 4 ).
3.2 Irradiation of Mice and Injection of Donor BM Cells
1.
Recipient male or female mice of at least 6 weeks of age (to be able to survive the irradiation) are irradiated with two doses of 650 rads (6.5 Gy) each (total irradiation of 1300 rads, 13 Gys). Regarding the various parameters of irradiation and veterinary care of irradiated animals see Notes 5 – 9 .
2.
3.
Transplanted mice should be allowed to recover for at least 1 month post-BMT.
4 Notes
1.
While preparing donor BM cells, if the amount of cells obtained from two femurs and two tibias is not sufficient, additional cells can also be harvested from the humerus, radius, and sternum.
2.
The cell-culture medium used for flushing of BM can be supplemented with antibiotics to avoid contamination but should not contain fetal calf serum. Alternatively, BM cells can be frozen and reutilized at a later time. For this purpose, donor BM cells are washed and resuspended in 1 ml of DMEM or RPMI medium containing 20 % fetal calf serum and 10 % DMSO (a cryopreservant) at a density of 107 cells/ml/vial. Cells are frozen in cryotubes at a temperature of −70 °C or in liquid nitrogen. When the BM cells need to be used again for transplantation, they should be thawed quickly in a water bath at 37 °C, washed several times with plain RPMI medium/sterile saline solution in order to get rid of the serum and DMSO, and resuspended in PBS or saline solution, prior to injection into the recipient/s.
3.
Before engraftment, it must be assured that the donor hematopoietic stem cells have no defect or deficiency in the homing molecules associated with their migration to the bone marrow, the lack of which would result in a failure of reconstitution. If the donor cells do have this deficiency alternatives such as orthotopic graft delivery (directly into the bone marrow) or wild-type BM cell supplements could be considered.
4.
Necrotic or apoptotic BM cells from donors will not survive the transplant. In this case the degree of cell death in the donor inoculum should be tested by measuring necrotic and apoptotic cell content using Annexin-V or propidium iodide staining, prior to being injected into the recipients.
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