Flow cytometry (FACS): Principle and experimental setup

Flow cytometry (FACS) is a method for the evaluation of cell-membrane proteins and intracellular proteins as well as peptides and DNA. The principle behind FACS is an antigen-antibody reaction, with the antibodies being fluorescently labelled. Quantification is being carried out with intercalating color-labels (without the antibody).

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The acronym FACS stands for Fluorescence Activated Cell Sorting. The term FACS is actually a brand name of Becton Dickinson (BD), but has gained popularity as the household name for flow cytometry. However, equipment and machinery for flow cytometry is being offered by various manufacturers under varying names.

The basis of a FACS analysis is a labelled (colored) suspensions of individual cells which passes a focused laser-beam. Capillary forces cause the cells to pass the flow-cell, where the labels are stimulated by the laser light. The emitted flurescent light from the flurophores, which are coupled to the antibodies, and the scattered-light are detected seperately.

LIGHT SCATTERING

The cells scatter a fraction of the light which is then detected by photomultipliers (light detectors). The amount of light measured correlates with the size of the cells and their complexity. Granulocytes for example reflect more light than smooth surfaced B- or T-cells, due to their rough surface texture and a larger amount of vesicles inside the cell. A measurement for the diffraction of light in a flat angle is the forward scatter (FSC), which depends on the volume of the cell. A measurement for the diffraction of light in a right angle is the so called sidewards scatter (SSC). It depends on the granularity, the size of the cells, the structure of its nucleus, and the amount of vesicles inside the cells. It is possible to distinguish blood-cells for example, without staining them, just looking at these two measurements (Fig. 1).

Fig. 1: Characterization of unstained cells using light scatter (Dot Plot). On the right hand side: large cells. On top: ganulary cells. Hence, large granulary cells (e.g. granulocytes) can be found on the top right, while small cells which are smooth can be found on the bottom left.

FLUORESCENCE MEASUREMENTS

Fluorescent colors can be measured concomittantly with the scattered light in flow cytometry. Only few cells produce flurescent light endogenously. Therefore color labels are used that bind to the cells. By using for instance the color substances DAPI and propidiumiodid, which intercalate in a cells DNA (between the base-pairs), the amount of DNA in a cell can be measured by determining the brightness of the cell. Also, fluorecently labelled antibodies can be used. Usually, the used antibodies target surface proteins (e.g. CD antigens; CD = Cluster of differentiation). The information density can be increased by using differently colored laser light, and filters. If the wavelengths of the emitted fluorescent light from the flurophores is distinguishable, several color markers can be used (multi-staining).

Fig. 2: Scheme of colored cells in a FACS measurement. The first image shows a positive CD80 and a negative CD14 cell. The antibody-coupled fluorochrome is being stimulated by laser light and emits fluorescent light of a certain wavelength (red). The light is being measured in flurescent channel 1. The second image shows a negative CD80 and positive CD14 cell Channel two measures fuorochrome light of another wavelength (green).

Using the equipment manufacturer's software, the end results of the measurements are presented graphically. The type of graphic can be chosen by the user. Most commonly Dot Plots and histograms are used. The above mentioned colorations could look something like this:

DOT BLOT

Fig. 3: Scheme of a Dot Plots for CD14 and CD80. Cells negative for both markers are on the bottom left. CD14-positive cells on the bottom right and CD80-positive cells on the top left. Cells positive for both markers are on the top right. The intensity of the fluorescence increases from left to right (x-axis) and from bottom to top (y-axis).

HISTOGRAM

Fig. 4:The two histograms present results of the surface markers CD14 (a) and CD80 (b) individually. The x-axis represents the intensity of the fluorescence, the y-axis the number of cells. The bar stands for the amount of positive cells.