Magnetic-activated cell sorting (MACS) is a highly specific cell sorting technique that integrates immunology, cell biology, and magnetic mechanics. Its high specificity comes from the specific recognition of antibody antigens. MACS technology has become the standard method of cell sorting, from laboratory to clinic, from small scale to large scale, from common cells to rare cells and complex cell subsets, from human and mouse cells to other cell lines. MACS technology provides a method of high-quality cell sorting in every laboratory.
The principle of cell separation by immunomagnetic beads method
The separation of cells by
immunomagnetic beads method is based on the fact that the cell surface antigen can combine with the specific monoclonal antibody conjugated with magnetic beads. In the external magnetic field, the cells labeled with magnetic beads by antibodies are adsorbed and remain in the magnetic field. The cells without this surface antigen are not labeled because they can not bind to the specific monoclonal antibodies conjugated with magnetic beads and do not stay in the magnetic field, so the cells can be separated.
Magnetic cell labeling modernThe most important thing in magnetic cell sorting using MACS technology is high-quality labeling. The labeling of positive cells should be enhanced as much as possible and the background staining should be weakened. There are two basic ways of magnetic labeling: direct labeling and indirect labeling.
1. Direct magnetic cell labeling
Direct labeling is the fastest and most specific magnetic labeling method. At present, there are a variety of directly labeled microbeads for sorting human, mouse, rat, and non-human primate cells.
2. Indirect magnetic cell labeling
Indirect magnetic cell labeling requires a combination of monoclonal or polyclonal antibodies and indirect labeling microbeads. Unbound antibodies, biotinylated antibodies, or fluorescein-labeled antibodies can be used as the primary antibody for labeling cells, and then anti-immunoglobulin beads, anti-biotin or streptavidin microbeads and anti-fluorescein microbeads can be used as the secondary antibody to magnetically label cells.
Almost any monoclonal or polyclonal antibody against any cells of any strains can be used for indirect labeling. Indirect labeling is mainly used in the following situations: when there are no direct labeling beads; multiple types of cells need to be sorted or removed with a mixture of several antibodies at the same time; indirect labeling has an amplified effect, so it can be used in sorting target cells with weak antigen expression; when homemade antibodies or ligands are used.
Sorting strategyrnThere are two basic sorting strategies: positive selection strategy and depletion strategy. The sequential selection strategy is the combination of the two basic sorting strategies or the combined use of multi-selected microbeads, so as to realize the sorting of cell subsets.
1. Positive selection strategy
In the positive selection, the target cells were magnetically labeled and directly sorted as positive marker components. The sorted cells do not need to remove magnetic beads and can be used for culture or follow-up operation immediately. The magnetically labeled target cells can be enriched 10000 times by this method. The advantages of positive selection strategy are high purity, high recovery rate, rapid and simple operation.
2. Depletion strategy
Depletion strategy is a method of removing non-target cells from the cell mixture after magnetic labeling, that is, unmagnetically labeled cells are target cells. The scope of application of the de-sorting strategy: removal of unwanted cells; lack of specific antibodies against target cells (such as tumor cells); no need for antibodies to bind with target cells, that is, cells are not stimulated (e.g. functional analysis of T cells, B cells, NK cells); part of the sequential selection.
3. Sequential selection strategy
The combination of two or more basic selection strategies is mainly used to sort cell subsets or to obtain highly pure and rare cells.
(1) Depletion followed by positive selection
The sorting of cell subsets can first magnetically label the non-target cells, remove them, and then carry out magnetic labeling and positive selection for the negative components. Scope of application: in the cell suspension, non-target cells also express antigens used for positive selection of target cells, so it is necessary to remove this group of non-target cells; if you want to sort very rare cells, remove non-target cells from the cell suspension first, and carry out positive selection on the basis of enriched cells, high purity target cells can be obtained.
(2) Multisort strategy
Multisort strategy is a technique for magnetic sorting of cells based on a variety of surface markers. In multiple sorting, first of all, the target cells were labeled with multi-selective microbeads, and the positive sorting of the first parameter was carried out. The cells are then incubated with a dissociation reagent, which can enzymatically dissociate the beads from the antibody. The positive cells were then sorted by magnetic labeling of antibody-bead complex against another cell surface marker. These cells can be sorted again for positive selection or depletion.
Important indexes of magnetic separation of cells.rnPurity and yield depend on the specificity of the monoclonal antibody conjugated to the beads and the size of the magnetic beads. However, large beads affect cell activity and cannot be detected directly by flow cytometry.
Advantages and disadvantages of magnetic beads of different sizes.rn1. Small magnetic beads (~50 nm)
Advantages:
(1) It is mild to the cells and does not affect the subsequent culture of the isolated cells.
(2) It can be detected directly by flow cytometry without affecting the scattered light.
(3) The magnetic beads occupy only a small part of the cell surface.
Disadvantages:
(1) A strong magnetic field is needed to separate cells.
(2) The separation speed is very slow and the yield is not high.
(3) A disposable separation column would be used instead of an ordinary test tube.
(4) The cost is high.
2. Large magnetic beads (1200-4500 nm)
Advantages:
(1) The technology is simple and the separation can be done in the test tube.
(2) It is easy to increase or decrease the number of involved cells.
(3) The separation speed is fast and the yield is high.
(4) The cost is low.
Disadvantages:
(1) It exerts mechanical pressure on cells and affects their biological activity, which is not conducive to isolation and culture.
(2) Cell purity is low. (3) It is easy to block the nozzle of flow cytometry.