Economical and Efficient Protocol of Isolating and Generating Bone Marrow-derived Dendritic Cells from Mouse. Procedures involving animal subjects have been approved by the Institution Animal Ethics Committee of Nanjing Medical University.One.Introduction. With the increase in tumor immunity research, the demand for DC cells is gradually increasing.
However, DCs are rare in all tissues. The traditional method of mainly inducing the differentiation of bone marrow into DCs is complicated and costly.Two. Isolation of bone marrow and preparation of BM cells We sacrifice mice and fix on mouse operating table.
Cut the skin of the left exposed muscles and femoral artery. Do not cut the femoral artery directly. Otherwise, it will cause heavy bleeding and contaminate the field of vision.
Cut all the muscles around the femur. Slowly stretch the lower limbs of the mouse outwards until prolapse of the femoral head and can be observed. Separate the lower limbs from the body.
Cut the hind leg to obtain a free and complete femur. Remove the muscles using gauze. Do not tear the muscles directly.
The femur of mice is fragile. Place the femur in 75%alcohol for two to five minutes.Three. Injection culture of BMDC Rinse the residual alcohol with PBS.
Clamp middle and bottom part of the femur with hemostatic forceps, and clamp lower end of the femur with another hemostatic forceps. The hemostatic forceps are applied literally to the femur and the femur is broken from epiphyseal line. Use one ml syringe to pierce the bone marrow cavity from the epiphyseal line.
Use complete medium two parts flush the bone marrow cavity until the bone becomes white. Collect flushing fluids. Supplementing the complete medium containing GM CSF/IL-4 to 24ml, and seed in a 6-well plate with 4ml per well.Four.Results.
After two days, replace all medium containing GM-CSF/IL-4. Half replaced complete medium containing GM-CSF/IL-4, on the fourth and sixth and eighth day. The number of cells reached a peak on the seventh day and then decreased gradually.
DC cells formed a large number of synapsis showing a typical mature DC cell morphology. Flow sub metric analyzes show that the ratio of CD11c imposed what 71%on the sixth day, wide ratio increase to 96.1%on the 10th day. The expression of CD11c, CD80 and MSC2, gradually increased with increasing cultivation time.Five.Conclusion.
In short, we established an economical and efficient protocol for isolating and generating bone-marrow-derived dendritic cells from mice, which only takes 10 minutes to separate bone marrow cells. A high number of high purity DCs was harvested after six to seven days of culture with 10ng per ml, GM-CSF, and IL-4.
