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Antibodies and flow cytometry

Antibodies add up quickly to the budget. The panels we design are complicated. Let me explain from the beginning. We use antibodies to conduct a lot of flow cytometry experiments.

What is flow cytometry?

Flow cytometry refers to the measurement of cells in liquid suspension. Cells flow through a laser that can measure their physical characteristics. We can also use that laser to measure biochemical characteristics such as extracellular or intracellular proteins (markers) by using fluorophores.

What are fluorophores?

Fluorophores are particular molecules that have the ability to emit light at particular wavelengths when excited. We can use these to attach them to other molecules such as antibodies. We can set the laser to hit the fluorophore at its particular excitation wavelength and measure its emission wavelength using special detectors. There is a wide variety of fluorophores available on the market for us to choose from.

Because we can fuse these fluorophores with antibodies it allows us to do two things:

  1. Have a selection of the same antibody in different fluorophore configurations
  2. Have a selection of different antibodies in the same fluorophore configuration

Now we can measure expression.

Expression?

Each antibody is designed to bind to a particular protein. These proteins are made (expressed) in different amounts according to the requirements of the cell or in response to certain stimuli. Some are more abundant than others. Depending on the cell type some can be present while others are not. This helps us distinguish the cells from each other. Using flow cytometry, the more amount of fluorescently labelled antibody binds to the cell the higher its fluorescent intensity so more of that protein is present in that cell.

How can you use this in science?

By designing a panel of antibodies that bind to different proteins and with each antibody having a different fluorophore attached, we can actually classify cells according to their expression of those proteins.

This is valuable information. It allows us to distinguish cells, use proteins as disease markers and even track changes over time and from person to person.

In a lab setting, it gives us information about cell manipulation and effects of treatments or experimental conditions. We can hypothesize e.g. that treating with drug X will alter protein Y in the cell type and check to see if that is the case or not.

Flow cytometry is routine work these days. When you need to design many different panels for a variety of experiments it is rather daunting and difficult to keep track of all the fluorophores and markers. I usually find this tool of great use. It is free and works well for the basic design of panels.

–Konstantin Knoblich

 

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