Common methods for cell sorting
Cell sorting is an unavoidable experimental procedure for a variety of research fields such as immunology research, stem cell research, cell**, oncology research, etc., and only when cells with specific properties are isolated from multicellular samples to achieve a certain level of purity, can they be subsequently analyzed.
Broadly speaking, cell sorting is divided into two broad categories of commonly used methods: density gradient centrifugation based on the physical properties of the cell, and methods based on immune recognition properties. However, the cells isolated in the former are of low purity, poor specificity, and unclear surface markings, and should be counted as cell isolation more strictly speaking. Methods based on immune recognition characteristics include fluorescence-activated cell sorting (FACS) and magnetic-activated cell separation (MACS).
Basic principles of flow and magnetic sorting
FACS was born thanks to the rise of flow cytometry, the basic principle of which is that when a droplet containing a single cell passes through the laser beam, the various fluorescein, fluorescent dyes and their physical and biological properties make the scattered light signal of the cell different, and the droplet is given a charge according to the signal difference, so that the path is deflected when passing through the high-voltage electrostatic field and falls into different collectors, so as to complete the sorting of the target cell.
Schematic diagram of the flow cytometry sorting principle.
The basic principle of MACS is that by binding immunomagnetic beads with specific antibodies to cells with corresponding antigens, some cells are given magnetic properties, and when the sample flows slowly through the magnetic field, these cells will be trapped on the magnet, while cells without this antigen are not magnetic and do not stay in the magnetic field, so as to achieve the isolation and purification of the target cells.
Schematic diagram of the principle of magnetic sorting.
Advantages and disadvantages of flow cytometry and magnetic separationPK
Both FACS and MACS have been widely used since their inception. However, under different experimental requirements, FACS and MACS have their own strengths, and the more suitable method should be selected according to the experimental purpose. So, what are the pros and cons of FACS and MACS?
The first is the difference in cost. Flow cytometry equipment is expensive, and the cost of operation and maintenance and employment is also high. Due to the harsh requirements for the operating environment, it is necessary to set up a separate room to be operated by a special person, and due to the complex operation of the flow cytometer, professional operators are required; Critical components** are expensive, and if they are damaged, repairs and replacements can be a costly expense. The magnetic sorting equipment is generally only a fraction of the flow cytometer, the operating environment is not high, the general laboratory can be carried out, and it is very easy to get started, and the technical requirements for the operator are not high.
The second is the effect on the cells. Because the cells need to go through high-speed flow steps in the flow cytometry process, FACs stimulate the cells greatly, so they have a great impact on the viability of the sorted cells, which is not conducive to subsequent operations. Magnetic sorting, especially with nanobeads, has a negligible effect on cells.
In terms of sorting efficiency, because the flow cytometry needs to ensure that the cells are single through the laser, the cell concentration of the sample should not be too high, otherwise the cells are easy to stick, so the sorting speed is about 10e4 cells per second, while the magnetic sorting can reach 10e7 cells per second. In addition, magnetic sorting is much higher than flow sorting for the volume of a single processed sample.
However, in terms of functionality, flow cytometry is superior to magnetic sorting equipment because it also has analytical functions. In terms of cell sorting purity, flow cytometry can achieve higher purity and perform multi-parameter sorting at the same time, but magnetic sorting can also improve the purity of target cells and achieve multi-marker sorting by multiple sorting. However, when cells need to be sorted according to intracellular indicators, magnetic sorting is not possible.
So flow sorting vs magnetic sorting, which one do you pick?
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