Development of hematopoietic cells in bone marrow and thymus is highly dependent on stromal cells for support. Perturbations of hematopoietic stromal cell function can lead to inefficient or even malignant blood production. Our lab is focused on understanding stromal cell regulation of hematopoiesis and how it can be manipulated for therapeutic benefits.
The advent of single cell sequencing has driven a cascade of recent advances in many research fields. This is also the case in our field where single cell resolution has enabled the identification of heterogeneous stromal cell populations in hematopoietic organs that were previously unknown. Our protocols are optimized to address this, and we hope they will enable more research on stromal cells to tackle critical questions in the field.
To begin, position the euthanized mouse on a surgical platform and open the chest cavity. Using surgical scissors, make a transverse incision below the rib cage, and cut ribs on each side of the mouse to the clavicle. Then cut the diaphragm to release the chest wall, and lift ribs with forceps to expose the thoracic cavity.
Hold the thymus with forceps and gently cut the connective tissue holding the thymus in place. To perform enzymatic dissociation of murine thymic tissue, place the clean thymus in a 15 milliliter tube cap, and use sterile surgical scissors to finally mince the tissue. Add two milliliters of murine dissociation cocktail to the tube.
Attach the cap containing the thymus pieces and invert the tube five times to resuspend the tissue. Wrap the tube cap in paraffin film. After incubation, place the tube in a rack and allow the tissue to settle.
Remove the supernatant and pass it over a 70 micrometer cell strainer into a 50 milliliter ice cold tube. Then add 20 milliliters of M199 with 2%FBS medium to the cell suspension. Centrifuge the tube at 500g for five minutes at 4 degrees Celsius.
After discarding the supernatant, resuspend the pellet in M199 and 2%FBS medium. To begin, take the femur, tibia, pelvis, and humerus bones isolated from a mouse. Use a scalpel to cut through the growth plates of long bones, removing the epiphysis.
Place it in a 10 centimeter dish with M199 medium plus 2%FBS on ice. Next, use a 28 gauge needle and a 10 milliliter syringe filled with M199 medium plus 2%FBS to flush the marrow into a 15 milliliter tube on ice. Place the flushed bone in the dish with the epiphysis.
Leave the 15 milliliter tube upright on ice until the marrow settles. Then remove the M199 medium plus 2%FBS. Add four milliliters of the B and M dissociation cocktail to the 15 milliliter tube, and incubate in a 37 degree Celsius water bath.
After incubation, collect the supernatant and filter through a 70 micrometer cell strainer into a cold 50 milliliter tube. Add two milliliters of fresh B and M dissociation cocktail to the remaining bone marrow pellet and return it to the water bath. Using a one milliliter pipette, vigorously pipette any remaining bone marrow pieces.
Collect all cell suspension in the same tube as before. Add 20 milliliters of M199 medium with 0.5%BSA, and two millimolar EDTA to the cell suspension. Afterward, centrifuge the cells at 500g for five minutes at 4 degrees Celsius.
Resuspend the pellet in 500 microliters of PBS with 0.5%BSA and biotin antibodies. Incubate on an orbital shaker for 10 minutes at room temperature in the dark. Next, vortex the magnetic beads thoroughly.
Add 25 microliters of the beads to the tube and incubate again on the orbital shaker. Place the tube in a matching magnet for two to three minutes and collect the supernatant in a fresh tube. Break the bones into smaller pieces in the 10 centimeter dish using the flat cap end of a 50 milliliter tube.
Filter the supernatant through a 70 micrometer cell strainer to isolate bone fragments. Cut the bone fragments in the cell strainer with scissors. Place the filter with the bone fragments on a 50 milliliter tube and open the filter with the scalpel.
With tweezers, transfer the bone fraction to five milliliters of murine B and M dissociation mix. Then place the tube horizontally in a 37 degree Celsius water bath with 120 RPM shaking for 30 minutes. After digestion, add 25 milliliters of M199 medium with 0.5%BSA and two millimolar EDTA.
Filter the supernatant containing stromal cells through a 70 micron cell strainer into a cold 50 milliliter tube.
