Mouse Bone Marrow Single Cell Suspension Preparation Process and Precautions

Preparation process of mouse bone marrow single cell suspension

1)   Kill mouse by cervical dislocation and soak it in 75% alcohol for 5 min. Prepare a sterilization tray on the ultra-clean table in advance (a sterile mask or gauze soaked with alcohol can be used instead), remove and spread the mouse on the sterilization tray.

2)   Remove the hindlimb bones of mouse in a sterile environment. Use ophthalmic forceps to carefully pinch the abdominal skin between the two hip joints of the mouse, carefully cut it with ophthalmic scissors, and separate the skin of the two lower limbs. The skin was cut down at the ankle and up at the hip joint to free both hind limbs of the mouse.

3)   Carefully peel off the muscle (The white tough tissue at both ends of the muscle is tendon. Muscle is mainly connected to the joint by the tendon and can be separated along the tendon). Cut off Femurs (femur bone) and Tibias (tibia) respectively. Cut off the cartilage at both ends and the red marrow cavity is exposed. Notice that the marrow cavity should be preserved as much as possible during this procedure.

4)   Take a 1 mL sterile syringe, draw 1 mL of PBS and gently insert it into the marrow cavity. Flush the marrow cavity to obtain bone marrow. Repeat 2~3 times to flush out most of the cells. After above steps, gently pipette the cells to disperse the cell clumps.

5)   Filter the rinse solution with a 200-mesh filter, collect the filtrate in a 15 mL centrifuge tube then centrifuge at 300 g for 5 min, discard the supernatant.

6)   Resuspend the cells in cell staining buffer, count the cells, and adjust cell concentration to 1×10⁷/mL.

Flow FSC/SSC diagram of mouse bone marrow cells

Precautions:

Bone marrow sample has obvious cell grouping, which can lyse red blood cells or not. If lyse red blood cells is needed, the procedure is as follows: add 1~2 mL (depending on the amount of cell pellet) 1× red blood cell lysate, gently blow off the cells, and stand at room temperature for 2 min. Add 10 mL of PBS to stop the lysis, centrifuge at 300 g for 5 min, and discard the supernatant.