To begin, mix the isolated murine bone marrow suspension with the bone spicule suspension. Pipette 10 microliters of the cell suspension onto a hemocytometer for counting. Centrifuge the cell suspension at 300 G for five minutes at four degrees Celsius.
Then resuspend the pellet in 100 microliters of FACS buffer. To stain the cells with antibodies for magnetic depletion, add FC block, CD45 APC, and TER-119 APC. Then wash the cell suspension with FACS buffer.
After centrifuging the remaining mixture, resuspend the pellet in 100 microliters of FACS buffer. To stain the cell suspension with microbeads for magnetic depletion, add mouse IgG and anti-APC microbeads. Incubate the mixture on ice for 20 minutes.
Wash the LD column with two milliliters of MACS buffer. Resuspend the cells in the buffer, then filter it through a five milliliter test tube with a 35 micrometer cell strainer cap. Next, set the LD column on the magnetic separator stand and place a five milliliter test tube under the column to collect the eluted material.
Pipette the cell suspension into the LD column and collect the negative fraction flow through in the test tube. Wash the column two times with one milliliter of MACS buffer and collect the eluent material in the same tube. Place the LD column over a new five milliliter test tube.
With a pipette, dispense three milliliters of buffer into the column, then flush out the positively labeled cells into a five milliliter test tube. Centrifuge both the positive and negative fractions, then resuspend the pellet in 100 microliters of FACS buffer. To stain the CD45 TER-119 negative fraction add one microliter of each antibody for every 10 to the sixth cells.
Place the suspension on ice for 20 minutes. Then wash the suspension with two milliliters of FACS buffer before centrifuging. Add one milliliter of the buffer to the pellet and pipette propidium iodide for live cell staining.
Finally, filter the sample through a 35 micrometer cell strainer into a five milliliter test tube before analyzing the cells on a multicolor flow cytometer. Arteriolar endothelial cells significantly expanded in the acute myeloid leukemia microenvironment with a concomitant loss in the sinusoidal endothelial populations. A small expansion in the mesenchymal stromal cells were seen at eight weeks of age.
