Introduction to the separation method of mouse spleen dendritic cells

Mouse spleen DC is the most studied lymphoid tissue DC characterized by constitutive expression of MHC class II molecules and CD11c. The cell population can be further divided into three groups: CD4+CD8-CD11b+DC mainly distributed in the marginal zone, CD4-CD8+CD11b-DC mainly distributed in the T cell region, and double negative DC----CD4-CD8+ CD11b+. Among them, CD8+DC expresses CD1d and DEC-205. We can choose different methods to sort the spleen DC according to the needs.

Mouse spleen DC can adhere to the surface of glass or plastic in vitro, but after 18-24h of culture, its adhesion ability is lost, thereby separating from macrophages. This method requires no special reagents and does not require density gradient centrifugation, which is simple and time-saving. However, the number of DCs obtained by this method is small, the purity is low, and the separated DCs are in different stages of differentiation, which cannot meet the needs of fine experiments.

CD11c is expressed on the surface of all known mouse DCs, so the CD11c antibody coated with magnetic beads is co-incubated with mouse lymphoid tissue or non-lymphoid tissue suspension and placed in a magnetic field. Cells bound to CD11c antibody will remain in the fraction. In the column, CD11c-positive DCs can be isolated after leaving the magnetic field. The method is simple and easy, and if properly operated, the purity of the cells can reach 95% or more. However, it is expensive to purchase the corresponding sorting equipment and reagents.

The surface of the DC expresses a variety of relatively specific surface markers. According to the needs of the experiment, a monoclonal antibody with specific surface markers can be used as a marker to identify the target cell population and perform automatic sorting by flow cytometry.
The method can perform cell sorting according to a plurality of markers, which makes up for the deficiency of MACS sorting, and the isolated DC cells have higher purity. However, the main problem currently existing in FCM sorting is that there are many types of DCs, and the development process and surface features are not fully understood. Therefore, when performing DC separation, it is necessary to select a suitable monoclonal antibody selection marker depending on the source of the specimen and the target cell. In addition, flow sorting must be carried out for flow sorting. The flow cytometer with sorting function is expensive and requires high technical requirements for operators.