Flow cytometry antibodies, kits, reagents

Chapter 1: Principles of the Flow Cytometer

Electrostatic Cell Sorting

A cell sorter provides the ability to separate cells identified by flow cytometry. 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. Droplets can be formed by using high-frequency (cycles/second, Hz) vibration of the nozzle at an optimal amplitude (in volts) over a period of time. This is typically created by a piezoelectric crystal.

There are two types of electrostatic sorters, which differ by where the particles are interrogated by the laser. 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. After the particles are illuminated at what is called the interrogation point, they continue down the stream. Data collected from the particles as they pass through the lasers at the interrogation point is sent to a computer, where the decision is made whether a given cell meets the criteria the user has defined for a desired particle. 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. One of the most critical parameters of sorting is to measure the distance between the point of interrogation and the exact point where the droplet breaks off. This distance is called the drop delay. When the cell gets to the last connected drop, the entire stream is charged at the nozzle. As the cell 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).

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. The droplets eventually pass through a strong electrostatic field and are deflected left or right based on their charge (Figure 6). Uncharged droplets pass into the waste.

Electrostatic flow sorting

Figure 6: Electrostatic flow sorting

The speed of flow 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 10,000–100,000 droplets per second. The stability of the break-off dictates the accuracy of the sorting.