FACS Guide : A Researcher’s Complete Guide to Fluorescence- Activated Cell Sorting
FACS or Fluorescence-activated cell sorting is a unique and specialized form of flow cytometry. After getting a tremendous response and need of more information from you all for the Flow cytometry and other concepts related to flow cytometry I decided to give a better picture on a more distinctive form of flow cytometry, i.e, FACS.
So here I shall throw light on the FACS Analysis and the types of lights used in this process.
FACS is a technique used for sorting a mixture that is heterogeneous in nature containing biological cells. The mixture is present in two or more containers and sorting is done of one cell at a time depending on the specificity of light scattering as well as its fluorescent traits. FACS is useful in the analysis and sorting of live cells as it provides the specific, intendedly fast and quantitative readings of fluorescent signals. This is done for individual cells with the help of physical separation of cells which are of any specific interest.
Here you will get a more clear picture of FACS and how we tend to do its analysis.
In a fast flowing liquid, the cell suspension is then determined in the center. With respect to the diameter, the cells are arranged which makes the separation large between the cells. The cells thus break into individual droplets owing to the stream which causes a vibration mechanism. In order to maintain the low probability of more than one cell per droplet, the system has to be adjusted in a precise way. Before the breaking of droplets, the fluorescent character of each cell is measured when it passes through the fluorescence station. The point where the stream breaks into droplets we are supposed to place an electrical charging ring at the same point.
FACS Image source: Sino biological
Prior to fluorescence intensity measurement, a charge is placed on the ring based on the separation, and on the droplet, the opposite charge is trapped as it breaks from the stream. Later the charged droplets are passed through an electrostatic deflection system. The electrostatic field causes the diversion of the droplets based upon the charge in different containers. However, there are other systems, in which the stream is directly applied with charge, and when the droplets break of the stream, they retain the same charges as of the stream. After the droplet break, the stream is again returned to the neutral phase.
To a mixture of cells a fluorescent molecule and an antibody specific for a particular cell surface protein is then added. The fluorescence is measured for the cells when they are passed through the laser beam.
Based on the tagging of the antibody using fluorescence the charging is given to the droplets be it negative charge or positive charge. According to their charge, a single cell containing a single cell is then detected by an electric field.
The cell of interested is first labeled with an antibody for a particular cell surface. To measure the fluorescence of each cell the antibody is coupled to a fluorescent dye and hence individual cells can pass through the laser beam. Small droplets are formed which contains a single cell and is given a positive or negative charge depending on whether the cell contains fluorescing. In order to ensure that each container has a homogeneous population of cells in each container, the cells are supposed to be tagged with a fluorescent antibody.
In the field of biochemistry, these homogeneous cultures may be used. Flow cytometry can be used to measure the DNA and RNA content of a cell.
Types of Lights used in the Flow cytometry Experiment
Image source: Antibodies Online
Forward scattered light moves along the light path and is inclined by a cell in the channel which means it is in the same direction as that of the original one. This forward scattered light is used to identify particle size which is detected by a sensor.
Side Scattered light is refracted by cells when it passes from the illumination source and the deviation is outside of the original path of light and the sensor is orthogonal to the original light path.
FACS Image Source: Antibodies Online
Fluorescent light is emitted after the excitation by a wavelength that is emitted by fluorescent molecules. Here it is specific to fluorescently label antibody only.
FACS Image Source: Antibodies Online
After the experimentation, comes the result. But are we clear how to analyze and interpret the FACS result?
How does the FACS data look like and how to analyze it?
Every single cell that is passed through the flow cytometer in the flow cytometry experiment is detected and will be tabulated as a clear event.
The types of light which were explained above is detected by the flow cytometer and is assigned its unique channel. Flow cytometry data plots every event independently and produces the signal which depicts the intensity of light in each of the channels for every distinct event.
This was how the result is extracted.
Now we will have a look at how we represent a Flow cytometry data.
This is done in two ways: Histograms which particularly measures a single parameter and then we have Dot-plots which measures two or three parameters concurrently on a two or three-dimensional scatter-plot.
Histogram plots the channel single in number on one axis and the number of events detected at the same intensity in another axis. The histogram gives the representation of a spike whenever the intensity is noted at any event.
Dot-plots have each event depicted as a single point on a scatter-plot. The intensity of different channels is represented along different axes. And we will get to know the result when the events are noted. All the events with similar light intensities will cluster together around the same region on the scatter-plot.
Dot-plots and Histograms have different advantages over each other and hence that can be considered while choosing the plots for the analysis of flow cytometry data. The smartness in choosing the best representation for your data surely makes a difference in the complete analysis and hence you can get a completely different story.
This was all about Fluorescence Activated Cell Sorting (FACS). I would be continuing ahead with some other important aspects involved in flow cytometry.
Let us make ourselves a pro in FACS!
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