Cell sorting is a technology to isolate and purify one or more cell population(s) from a heterogeneous single cell suspension. The sorting process is based on predefined criteria like cell size, shape, morphology and surface or intracellular protein expression. Flow cytometric cell sorting is used in a wide field of applications like physiological research, protein engineering, cell engineering and for medical purposes like cell therapy (autologous adoptive cell transfer "ACT") or transplantation.

The most widely used type of sorting is electrostatic cell sorting. The fundamental process of this technique is the formation and charging of droplets. Most cell sorters use a piezoelectric device to form droplets by the application of an acoustic wave in the flow cell of the instrument.  By that means droplet formation is extremely fast and stable with a predictable rate. 

For most electrostatic cell sorters, cells are transported through the system in a stream of sheath fluid. Based on the principles of hydrodynamic focusing, cells are lined up like beads on a string in direction of the flow. At the interrogation point cells are hit by laser light and the resulting optical signal is collected and processed by optical and signal processing systems. When a target cell is detected, a charge is applied to the droplet that contains the cell of interest. All droplets then pass through an electric field created by deflection plates: charged droplets are deflected toward collection tubes as side streams, while uncharged droplets continue into the waste.

The Core Facility operates with a BD FACS ARIA III and BD FACS Discover S8 which are both charged droplet sorters. In these machines, the cells are forced through an optically gel-coupled cuvette flow cell to interrogate with the laser(s). This cuvette-based interrogation gives a excellent optical resolution to distinguish dimly positive populations from background fluorescence. In addition, scatter performance is optimized for resolving lymphocytes, monocytes and granulocytes.

One main difference between the two machines is, that the BD FACS ARIA III as a high-speed sorter manages to sort up to 20000 events per second using the 70µm nozzle, while the BD FACS Discover S8 possesses the 85µm nozzle managing to sort only 10000 event per second. Therefore, the ARIA III should be used for sorting large numbers of cells. However, fast sorting comes with the disadvantage that high sheath pressure and a small nozzle diameter could potentially harm sensitive cells, like B-cells or dendritic cells.

The BD S8 belongs to the new class of spectral flow cytometers and cell sorters. With the unique SpectralFX technology, the in-depth detection of rare population in a complex panel of up to 50 markers can be analyzed and sorted. By using the spectral unmixing algorithm, autofluorescence of difficult samples like skin or brain can be extracted to achieve optimal sorting results. On top, the state-of-the-art CellView technology record images of each cell and enables the identifcation of additional morphological features of the cells.

The cell sorters can be operated with different nozzle sizes. The size of the nozzle depends on the dimensions of the cells to be sorted. As a general rule of thumb: the cell size should not exceed one-fifth of the nozzle diameter. The table below illustrates the sorting conditions related to the different nozzle sizes.

Using the wrong nozzle size causes fanning of the side streams, poor sort purity and nozzle clogging.

Cell sorting takes time. The time that is needed to sort the desired number of your target cells is dependent among others on the nozzle size, the sort mode (enrich versus purity), percentage of target cells in the sample, and the sample itself. The following table should help to estimate the sort time.