Mouse Bone Marrow Single Cell Suspension Preparation Procedures and Precautions
Preparation of mouse bone marrow single-cell suspensions
1. Dislocate the cervical vertebrae and soak the mice in 75% alcohol for 5 min. prepare a sterilized tray on the ultra-clean table in advance (can be replaced by a sterile mask or a gauze soaked with alcohol), remove the mice and lay them on the sterilized tray.
2. Remove the hind limb bones from the mice in a sterile environment. Carefully pinch the abdominal skin between the two hip joints of the mouse with ophthalmic forceps, carefully cut it with ophthalmic scissors, and separate the skin of the two lower limbs. Cut the skin downward at the ankle and upward at the hip joint to free both hind limbs of the mouse.
3. Carefully peel off the muscles (the white tough tissue at the ends of the muscles are tendons, which can be separated by following the tendons; the muscles are mainly attached to the joints by the tendons), cut off the Femurs (femurs) and Tibias (tibia) separately, and cut away the cartilage at both ends to expose the red bone marrow cavity. Note that as much of the marrow cavity as possible should be preserved during this procedure.
4. Take a sterile 1 mL syringe, aspirate 1 mL of PBS, gently insert it into the bone marrow cavity, and flush the marrow cavity to obtain bone marrow. Repeat 2~3 times to flush out the majority of cells. After rinsing, gently blow the cells with a pipette to disperse the cell mass.
5. Filter the rinsate through a 200-mesh filter, collect the filtrate in a 15 mL centrifuge tube, centrifuge at 300 g for 5 min, and discard the supernatant.
6. Cells were resuspended with cell staining buffer, cells were counted, and the cell concentration was adjusted to 1×107/mL.
Flow FSC/SSC plot of mouse bone marrow cells
Precautions:
Bone marrow samples with obvious cell clustering may or may not be lysed for erythrocytes. If erythrocytes are to be lysed, add 1~2 mL (depending on the amount of cellular precipitation) of 1× erythrocyte lysate, gently blow the cells apart, and let them stand at room temperature for 2 min. add 10 mL of PBS to terminate lysis, and centrifuge at 300 g for 5 min.
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