This method can help answer a few questions in the method of malignancy field about characterization of a cell that initiate a malignancy. The main advantage of this technique is that it provides a foundation for the functional analysis of these cancer stem cells. The implications of this technique extend to where the therapy of myelodysplastic syndromes or MDS, as it can aid in the identification of the disease initiating cells.
To prepare the recipients for adoptive transfer, one week before the transplant provide sterile water supplemented with 100 milligrams per liter of ciprofloxacin to con genic recipient ANIMALS for seven days in a specific, pathogen free animal facility. On the seventh day, have a trained, certified cesium 17 operator set the cesium source instrument to deliver 70 centigrayS of total body gamma irradiation per minute. Place 10 mice in a custom-designed holder.
Insert the holder into the irradiator chamber, deliver 9 grays total body irradiation in 13 minutes. Then return the animals to their cages. The next morning, spray 70 percent ethanol over an entire two to six moth old C57 black six mouse and use scissors to remove the skin from the pelvis to the ankles.
Use forceps with the scissors to remove the femora and tibiae. Cleanly trim the muscles from the bones. Cut the proximal and distal ends for the tibiae and pick up one tibia with forceps.
Insert a three milliliter syringe equipped with a 27 gage needle into the bone marrow cavity and flush the bone marrow into a 14 milliliter round bottom tube containing 2.5 milliliterS of Hanks buffered saline solution supplemented with 2%fetal bovine serum or HF2. When all the marrow has been collected, aspirate the tissue several times through the 20 gage needle tip to disperse the marrow mass into a single suspension for counting. To label the bone marrow nucleated cells, or BMNC, for flow sorting, collect the cells by centrifugalization and re suspend the pellet at a one times ten to the seventh BMNC per 90 microliters of HF2 concentration.
Next, add ten microliters of a biotinylated, anti-lineage antibody cocktail to the cell suspension for 20 minute incubation at 4 degrees Celsius. Followed by a centrifuge wash in three milliliters of PBS. Re suspend the pellet in 100 microliters of HF2 per one times ten to the seventh cells.
Secondarily label the cells with two microliters of APC conjugated anti biotin antibody. After 20 minuets at four degree Celsius, protected from light, wash the cells in three milliliters of fresh PBS. Re suspend the pellet in HF2 supplemented with 0.2 micrograms per milliliter of propidium iodide on ice.
Now calibrate the flow cytometry cell sorter using unstained cells to optimize all of the photomultiplier tubes, scatter, and fluorescence parameters within the linear range of each detector. Acquire three samples of 10, 000 cells for the unlabeled, PI labeled and anti lineage APC labeled cells. At the end of the sort, analyze 1, 000 cells from each sample to confirm the purity and viability of the sorted cells.
Spin down the samples in the centrifuge. Then re suspend the pellets in 0.5 milliliters of HF2 for counting. For adoptive transfer of the sorted cells, mix 100 microliters of two times the to the fifth wild-type BMNC from a con genic recipient animal in sterile HF2.
With 100 microliters of two to five times ten to the fourth the sorted donor BMNC of interest in sterile HF2 in a micro centrifuge tube per recipient animal. Load one one milliliter syringe, equipped with a 28 gage per recipient with 200 microliters of cells. Then, place the recipient mice under a heat lamp to induce tail vein dilation.
Deliver the cell mixtures to each lethally irradiated recipient animal via the tail vein. As stem cell self-renewal is confined to lineage negative wild-type BMNC, lineage negative, low lineage positive, and high lineage positive cells are sorted and transplanted into wild-type recipients to determine the self renewal capacity of each cell population. Adoptively transplanted donor trans genetic BMNC from MDS model animals can be distinguished ex vivo by their CD45.2 cell surface marker expression.
By 16 weeks after transplantation of lineage negative BNMC, or unsorted BMNC from transgenic MDS donor animals, the transgenic cells out compete the wild-type cells as evidenced by an increasing percentage of CD45.2 positive donor cells, observed in the peripheral blood of the lethally irradiated recipient animals. Here cells from a leukemic transformation from a mouse transplanted with transgenic MDS cells are shown. Note the presence of numerous blasts and immature forms.
While attempting the procedure, it is important to remember to limit the time of ex vivo manipulation, as it can place undue stress on the cells, resulting in cell death or functional loss. After it's development this technique paves the way for the researchers in the field program, for putting in malignancy to explore the source of the disease in genetically engineered mice.
