Cell Sorting
Cell sorting is a powerful tool for basic and clinical research because individual cells can be isolated from heterogeneous sample and used for downstream analysis or therapeutic applications. Flow cytometry sorters allow physical separation of cell subpopulations from heterogeneous populations with a high degree of purity. There are two kinds of sorting mechanisms, the commonest method is by electrostatic deflection of charged droplets, while the other method use piezo-electric devices to sort the cells mechanically.
Sorting by Deflection of Charged Droplets
Most of the high speed cell sorters use the electrostatic cell sorting method, which is based on the electrostatic deflection of charged droplets. In this method, the cells are injected through a nozzle to form a stream of regular droplets by applying a vibration to the nozzle. Then, these droplets pass through one or more laser beams and are simultaneously charged by a charging electrode. Droplets can be deflected from the mainstream according to their given charges. The positively charged droplets are deflected toward a platinum plate with negative charge, the negatively charged droplets are deflected toward the positively charged platinum plate, and the uncharged droplets are collected into a waste container.
For precise sorting, it is very important to adjust several parameters, including:
- Nozzle vibration, conditioned by the drop drive frequency, the amplitude level, and the particle velocity.
- Time taken by the instrument to measure a particle’s signal and reset to measure the next particle (i.e. the time required to analyze one particle)
- Distance between the laser beam interception of the cell and the break-off point, where the stream beaks into droplets
Mechanical Sorting
In some sorters, separation of the desired population is achieved by using a piezo-electric device to deflect the cell out of the main stream. When cells pass through the laser beam, the system determines whether each cell belongs to the selected population defined by boundaries in the cytogram. If the cell is identified as a cell of interest, it will be captured by the catcher tube and collected into a tube or into a concentration module, otherwise it will be dispatched into the waste tank. The operator can choose the purity level of the sort among three levels.
The advantage of electrostatic sorting is the possibility to sort cell subpopulations at high speed. However, it is not suitable for sorting samples that have been treated with toxic substances because of the generation of aerosols. The high pressure produced in electrostatic sorting can also damage the sorted cells. Piezo-electric cell sorters have the advantage that they are very stable once they have been set up. The main drawback is that only one population of cells be sorted at a slow speed. Nevertheless there is no aerosol, so it is safe to sort samples which have been treated with toxic substances.
References
- Cossarizza A. et al.; Guidelines for the use of flow cytometry and cell sorting in immunological studies. European Journal of Immunology, 2017, 47(10): 1584-1797.
- Ibrahim S. F. et al.; Flow cytometry and cell sorting. Adv Biochem Eng Biotechnol, 2007, 106: 19-39.
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