Electrostatic Cell Sorting in Flow Cytometry

Author: Mike Blundell | Reviewer: Chloe Fenton

Electrostatic cell sorting is a flow cytometry technique that separates cells by charging droplets containing target particles and deflecting them using an electric field. It enables high‑purity isolation of specific cell populations for downstream analysis such as genomics, proteomics, or cell culture.

Overview of Electrostatic Cell Sorting

A cell sorter (for example, the Bio-Rad S3e Cell Sorter) provides the ability to separate cells identified by flow cytometry. Droplet-based cell sorters first analyze the particles but also have hardware that can generate droplets and a means of deflecting or directing wanted particles into a collection tube.

Droplet Formation in Electrostatic Cell Sorters

Droplets can be formed by using high-frequency (in cycles/sec, Hz) vibration of the nozzle at an optimal amplitude (in volts) over a period of time. This is typically created by a piezoelectric crystal. As with cell analyzers, sorting can now be performed using conventional and full-spectrum flow cytometers.

Types of Electrostatic Cell Sorters

There are two types of electrostatic sorters, which differ in where the particles are interrogated by the laser beam. Sense-in-air sorters illuminate particles as they exit the nozzle and enter the stream. In cuvette sorters, particles are illuminated in a quartz cuvette before they enter the stream.

Optical Interrogation and Sorting Decision

After the particles are illuminated at the interrogation point, they continue down the stream. Data collected from the particles as they pass through the laser beam, at the interrogation point, are sent to a computer, where the decision is made whether a given particle meets the criteria the user has defined for a desired particle.

Droplet Break-Off and Drop Delay

As the particle continues to travel down the stream, the stream eventually breaks into droplets and the particle of interest is captured in a drop. To prevent the break-off point happening at random distances from the nozzle and to maintain consistent droplet sizes, the nozzle is vibrated at high frequency.

One of the most critical parameters of sorting is to measure the time between the point of interrogation and the exact point where the droplet breaks off. The time and therefore the distance is called the drop delay.

Electrostatic Charging and Droplet Deflection

When the particle gets to the last connected drop, the entire stream is charged at the nozzle. As the particle of interest-containing drop breaks off, the drop becomes charged. The droplet then passes through an electrical field and is deflected into a tube or plate. Uncharged particles pass into the waste (Figure 6).

Sorting Speed and Accuracy

The speed of cell sorting depends on several factors, including particle size and the rate of droplet formation. A typical nozzle is 70–130 µm in diameter and can produce 10,000–90,000 droplets per sec. The stability of the breakoff dictates the accuracy of the sorting.

Applications of Electrostatic Cell Sorting

Cell sorting is commonly used to isolate cell populations and single cells for downstream experiments analyzing DNA, protein, or cellular function. Purifying cells based on markers such as CD34 in hematopoietic stem cells or viability is often used, as is selecting cells, either in populations or single cell cloning, for further culture.

Fig. 6. Electrostatic flow sorting. A, optical interrogation and light collection; B, stream partitioning into droplets; C, stream and droplet charging as the target particle passes through the break-off;
D, droplet deflection through an electrostatic field; E, uncharged droplets pass into the waste.