Flow Cytometry FACS Explained For Beginners

Опубликовано: 30 Октябрь 2022
на канале: Lucas Learns
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Flow cytometry uses light scattering caused by cells in a sample which are passed through a laser beam. This light scatters in the forward direction as well as the side direction and is called forward scatter, and side scatter respectively. Forward scatter is proportional to the size of the cell, while side scatter is proportional to the complexity of the cell. Both the forward and side scatter gets detected and converted into an electric pulse, which is directly proportional to the amount of scattered light. By combining these two signals into a graph, we get a so-called dot-plot which looks like this. Now this is just the most basic flow cytometry graph available to us, and we can get even more information about the different cells by using a process known as flow cytometry FACS, which stands for “fluorescence-activated cell sorting.

Let us examine how FACS differs from normal flow cytometry. By utilizing highly specific antibodies labeled with fluorescent conjugates, FACS analysis allows us to simultaneously collect data on, and sort a biological sample by a nearly limitless number of different parameters. An antibody specific for a particular cell surface protein is associated to a fluorescent molecule and then added to a mixture of cells. This results in the fluorescent labeled antibodies binding to any cells containing the surface protein which it is specific for. In other words, if we now detect that fluorophore later, we know that a cell containing that surface protein is present in the sample.

In the same manner as in regular flow cytometry, the data from forward-scatter and side-scatter is collected but now in addition, this fluorescent signal data is collected as well. This is done using multiple detectors as well as dichroic mirrors. Here we can see that as the light from the laser excites the fluorophores on the cells, they reemit light which can be detected. Depending on which combination of detectors pickup the light we know what fluorophore has been excited.

Now, let us examine how the machine can sort the cells with the help of this data:

A vibrating mechanism causes the stream of cells to break into individual droplets, each droplet containing a single cell. Each cell then gets analyzed based on the forward and side scatter as well as the fluorescent signal data. The cell sorting machine then imposes an electrical charge on each of these droplets, depending on a set parameters predetermined by the operator. Then the cells will be sorted by charge, with the help of electromagnets, into separate vessels upon exiting the flow chamber. In this manner we can for example specify that we want smaller than x cells in one vessel, and larger than x cells in another.


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