The described protocol allows for the phenotypic analysis of bone marrow hematopoietic stem and progenitor cells and bone marrow stromal cells. It can be used to study perturbations to these populations in different settings. Compared with other previously described methods, this technique allows for the phenotypic analysis of hematopoietic cells, specifically in the two functional marrow areas, endosteal and central bone marrow, as well as bone marrow stromal cells.
To begin, place the euthanized animal with the belly up on a Petri dish and spray with 70%ethanol. Make a cut above the abdomen using scissors and pull away the skin all the way to the ankles. Grab one of the legs and cut at its bottom to separate it from the animal using scissors.
Then remove the foot from the leg by cutting at the ankle and place the leg in ice cold PBS 2%FBS. Next, grab the hip bone using tweezers and cut behind it to separate it from the animal using scissors. Place the hip bone in ice cold PBS 2%FBS.
After placing the leg and hip bone in a Petri dish, clean the bones using a sterile scalpel. Then separate the tibia and femur by cutting at the knee with the scalpel and place the clean bones in ice cold PBS 2%FBS. Place the femur or tibia in a mortar with ice cold PBS 2%FBS and crush the bone by gently pressing it against the wall of the mortar using the pestle.
Next, pipette the resulting cell suspension up and down using a 10 milliliter pipette to homogenize and transfer it into a 50 milliliter tube through a 40 micrometer cell strainer placed on top of the tube. Rinse the mortar with some more PBS 2%FBS and transfer the solution into the same 50 milliliter tube through the same 40 micrometer cell strainer. Centrifuge the 50 milliliter tube at 500 G for five minutes at four degrees Celsius.
After discarding the supernatant, resuspend the cell pellet in five milliliters of RBC lysis buffer at room temperature by pipetting up and down several times. Following a two-minute incubation at room temperature, add 15 milliliters of PBS 2%FBS and centrifuge the 50 milliliter tube. If the cell pellet does not look reddish, discard the supernatant, resuspend the pellet in 200 microliters of PBS 2%FBS and transfer the cell suspension into a well of a 96-well V bottom plate for staining.
After crushing the bone as demonstrated previously, cut the tip of a P1000 pipette using scissors and use it to transfer all the content of the mortar into a 50 milliliter tube. Then centrifuge the 50 milliliter tube at 500 G for five minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in three milliliters of collagenase IV and this dispase-II solution.
Incubate the tube at 37 degrees Celsius for 40 minutes. Top up the tube to 25 milliliters with PBS 2%FBS and vortex to mix. Next, transfer the content to a new 50 milliliter tube through a 100 micrometer cell strainer.
Then add 15 milliliters of PBS 2%FBS to the old 50 milliliter tube and transfer the solution into the new 50 milliliter tube through the 100 micrometer cell strainer. After centrifuging and discarding the supernatant, resuspend the cell pellet in five milliliters of RBC lysis buffer by pipetting up and down several times with a pipette. Following the two-minute incubation at room temperature, add 15 milliliters of PBS 2%FBS.
Again, centrifuge the tube and if the cell pellet does not look reddish after discarding the supernatant, resuspend the pellet in 200 microliters of PBS 2%FBS and transfer the cell suspension into a well of a 96-well V bottom plate for staining. Then centrifuge the plate at 500 G for three minutes at four degrees Celsius. Place the femur on a Petri dish and cut the ends of the bone using a sterile scalpel.
Then flush the central part of the bone by passing 100 microliters of PBS 2%FBS through the inside of the bone once from each side using a no dead volume 0.5 milliliter insulin syringe and collecting the solution in a microcentrifuge tube containing one milliliter of PBS 2%FBS. Subsequently transfer the cell suspension into a 50 milliliter tube labeled as flushed bone marrow containing four milliliters of PBS 2%FBS. Crush the ends of the bone in a mortar with ice cold PBS 2%FBS by gently pressing them against the wall of the mortar using the pestle.
Once the cell suspension is homogenized, transfer it into a 50 milliliter tube labeled as crushed bone marrow through a 40 micrometer cell strainer. At last, rinse the mortar with some more PBS 2%FBS and transfer the solution into the same tube. Flow cytometry analysis of hematopoietic stem cells and multipotent progenitors was performed in total bone marrow in a healthy young adult C57BL6J mouse.
In the analysis of stromal cells, endothelial cells, and mesenchymal stem cells in total bone marrow, mesenchymal stem cells can be readily identified based on leptin receptor expression and endothelial cells can be identified based on the expression of CD31 and SCA-1. Flow cytometry analysis of endothelial cells in total bone marrow showing the discrimination between arteriolar and sinusoidal endothelial cells based on ICAM1. The quantification of arterial and sinusoidal bone marrow endothelial cells in central and endosteal bone marrow tissue shows that arterial bone marrow endothelial cells are more abundant in endosteal regions, while sinusoids are more frequent in central bone marrow areas.
When obtaining flushed bone marrow, the volume or number of times indicated to pass PBS 2%FBS through the inside of central part of the bone should not be exceeded. Combining the described method with functional assays of the cell populations phenotypically analyzed would provide further valuable information about the potential perturbations to these populations occurring in the studied conditions. The described protocol allows the phenotypic analysis of hematopoietic cells differentially in endosteal and central bone marrow, enabling researchers to investigate perturbations to hematopiesis potentially occurring specifically in these bone marrow locations.
