Team:TU Eindhoven/Background/FACS

FACS: Fluorescence Activated Cell Sorting

To analyze the general system, fluorescence activated cell sorting (FACS) is used. FACS is an extended version of flow cytometry that enables separation between cells with different fluorescence characteristics. (Figure 1) This is particularly useful in the analysis of samples containing mixtures of biological particles.

In flow cytometry, the cells in the sample are first hydrodynamically focused, which means that a stream of sheath fluid is applied in such a way that the cells pass the lasers one by one. The lasers excite the fluorescently labelled cells to emit light at varying wavelengths, which will be collected by several fluorescence detectors. All these fluorescence detectors contain filters, so that each detector is capable of detecting the fluorescent intensity of a certain range of wavelengths. In this way, cells that have been fluorescently labelled differently can be distinguished.

Besides fluorescence detectors, also scatter detectors are placed around the stream. In line with the light beam a forward scatter detector (FSC), and perpendicular to the stream a side scatter detector (SSC) have been placed. These detectors determine the cell volume and inner complexity, respectively, and are used to distinguish between cells, particles and clumps of cells.

After passing the laser beam, the cells in the sample proceed to a nozzle. The tip of the nozzle is coupled to a transducer that causes vibration. This vibration is applied to obtain a stable break-off point and size of the droplets. By measuring the droplet delay between the point of measurement and the point of break-off, it is possible to independently charge individual droplets as they break away from the solid stream. The droplets can either be given a positive, negative or no charge, based on their fluorescence characteristics. Separation between independently charged droplets is then caused by a static electrical field created by two oppositely charged plates.

Our Project

For our project it has to be analyzed whether DBCO functionalized polymers can be attached to the azide-functionalized outer membrane proteins. In order to do this, first only the flow cytometry functions of FACS will be used. (Figure 2)

First, small DBCO functionalized fluorophores and fluorescently labelled antibodies will be applied to the system, in order to determine whether the azide functionalized outer membrane proteins have been expressed successfully. For this, the fluorescence detecting function of FACS will be used.

Secondly, it also has to be determined whether the larger DBCO functionalized molecules can be applied to the system. Unfortunately, these molecules cannot be fluorescently labelled. Therefore, the forward scatter detecting function of FACS will be used, in order to determine a change in size before and after reaction with the larger molecules. Depending on the time left, also cell sorting experiments will be conducted.

Figure 2. A schematic overview of the functions of FACS we will mainly use to analyze our general system.

Bibliography

[1] Macey, M. G., & Davies, D. (2007). Cell sorting by flow cytometry. Flow cytometry principles and applications (pp. 257-276). Totowa, NJ: Humana Press.