Our research aims to explore how the bone marrow's changing environment influences tumor growth. We developed a flow cytometry protocol to analyze the non-immune cells in bone marrow and bone tissues, focusing on marine hematological malignancies. This method is adaptable for studying bone marrow in various mirroring models.
Our study focuses on isolating and identifying alterations in bone marrow stromal populations during cancer progression. Understanding their impact on disease onset and advancement could pave the way for new treatments that target these cancer facilitating changes in the bone marrow stromal cells. Our protocol uniquely combines mechanical and enzymatic digestions, magnetic depletions, and flow cytometry to delineate the bone marrow microenvironment cellular makeup.
It stands out by employing magnetic separation and incorporating cells from both bone and marrow, offering a more comprehensive representation of this microenvironment. To begin, remove the tissue from the bones of the euthanized dissected mouse. Place the cleaned bones into a new six-well plate containing FACS buffer placed on ice.
Transfer the bones into a mortar with two to five milliliters of FACS buffer. Grind the bones in a circular motion until the bone marrow is released. With a three milliliter syringe, pull up and flush down the bone marrow to homogenize it.
Pull the homogenate into another syringe, then filter it through a 70 micrometer cell strainer into a 50 milliliter tube. Rinse the tissue chunks from their filter back into the mortar with FACS buffer before homogenizing a second time. Filter the rinsed tissue as before.
Next, rinse the remaining bone spicules back into the mortar with FACS buffer. Then flush the pieces into a 15 milliliter tube to ensure maximum yield. To digest the bone marrow, first centrifuge at 300G for five minutes at four degrees Celsius.
Resuspend the bone marrow pellet in two milliliters of digestion mixture. Transfer the mixture to a 15 milliliter tube, then incubate it at 37 degrees Celsius for 45 minutes on a rotator. Now add 10 milliliters of FACS buffer to stop the enzymatic digestion.
Filter the mixture through a 70 micrometer cell strainer into a 50 milliliter tube. Centrifuge the suspension at 400G for seven minutes at four degrees Celsius. Resuspend the pellet in one milliliter of RBC lysis buffer.
Next, add 10 milliliters of FACS buffer to halt the lysis. Then filter the mixture as before into a 50 milliliter tube. Lastly, centrifuge the mixture at 300G for five minutes at four degrees Celsius.
After discarding the supernatant, resuspend the pellet in 100 microliters of FACS buffer. For the digestion of bone spicules, first vortex the bone spicules. Decant the supernatant and retain the bones at the bottom.
Next, resuspend the spicules in one milliliter of the bone spicule digestion mixture. Incubate the tube on a rotator for 60 minutes at 37 degrees Celsius. Finally, stop the digestion with 10 milliliters of FACS buffer.
Filter the mixture into the 50 milliliter tube containing RBC-lysed and digested bone marrow. 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 300G 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 micro beads for magnetic depletion, add mouse IgG and anti-APC micro beads.
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 eluded 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 eluate 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. Sustain the CD45 TER-119 negative fraction, add one microliter of each antibody for every 10 to the 6 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.
