How to extract mouse bone marrow cells?

summary

The sternum, ribs, vertebrae and other flat bones of animals as well as the two ends of long bones of limbs are the main places of hematopoiesis. Bone marrow can reflect the state of hematopoiesis. Therefore, the use of bone marrow puncture method to check and observe the changes in the number and quality of bone marrow cells is of great value for the diagnosis, treatment effect, observation and theoretical research of the disease after animal experiment. How to extract mouse bone marrow cells?

How to extract mouse bone marrow cells?

1. The mice (C57 mice) were killed by cervical dislocation. After soaking in 75% alcohol for 3-5 minutes, the mice were placed on a disinfection tray on the super clean table. Aseptic extraction of mouse limbs bone, with ophthalmic forceps carefully pinch the abdominal skin between the two hip joints of mice, with ophthalmic scissors carefully cut the skin, and separate the skin of the two lower limbs, cut down at the ankle, cut up at the hip joint, so as to free the two lower limbs of mice.

Carefully peel off the muscle, cut off femurs (thigh bone) and tibias (tibia), cut off the cartilage at both ends to expose the red marrow cavity. Pay attention to cut the marrow cavity as little as possible. Wipe the muscle on the bone with gauze. Take a 1ml sterile syringe, suck 1ml HbSS solution from each syringe, and gently bend it with sterile syringe. Insert the bone marrow cavity gently and wash the bone marrow cavity to obtain bone marrow. Generally, wash the bone marrow cavity twice with 2-3ml medium, and most of the cells can be washed out.

Filtration: filter with a 2oo mesh filter, insert the center of the filter into the centrifuge tube at 25px, then suck the bone marrow fluid with a syringe, and slowly inject the bone marrow fluid into the centrifuge tube through the filter to remove the residue. Centrifugation: place the centrifuge tube in the centrifuge for 10 minutes (1350 R / min) to remove the supernatant, add 1 ml of erythrocyte lysate ACK, stand for 3 minutes, and then add 9 ml HbSS solution for centrifugation for 10 minutes, discard the supernatant.

matters needing attention

Bone marrow cells were washed twice with HbSS solution and centrifuged at 1350 R / min for 10 minutes at room temperature. The living cells were counted, and the cell concentration was adjusted to 1 × 106 cells / ml with culture medium. The bone marrow cells were washed twice with HbSS solution, centrifuged at 1350 R / min at room temperature for 10 minutes. The living cells were counted, and the cell concentration was adjusted to 1 × 106 cells / ml with culture medium. The culture medium was absorbed and added to the precipitate. The 24 well plate was used for plate culture, and each well was 1 ml