1st August, 2017.

Flow Cytometry

Flow Cytometry is a technique used to identify biomarkers on cell surfaces and intracellular molecules. Most flow cytometers use fluorescently labelled antibodies to differentiate between cells expressing different markers.

Initially the cell sample is suspended, stained with a fluorescent label and washed to remove any non-specific binding. The suspension is introduced into the cytometer and the cells then undergo hydrodynamic focusing, ensuring that they pass the sensor in single file. The cells flow past a laser beam and there are two methods to detect cells.

The first is that the forward and side scatter of light is detected from each cell as it passes through the laser. The direction of light scattered by each cell directly correlates with the size of the cell and its granularity.

The second is through fluorescence which is emitted from the stained cells. When stained cells move past the laser, the fluorophore on the cell is excited. This fluorescence is reported at a different wavelength to the laser. Positive detection is shown through an increase in the number of events at the fluorescence of the label.

Aptamer, Flow Cytometry, Data, Stain, Antibody Alternative

Figure 1: Aptamers used to stain two cell strains. Aptamers were raised to cell strain A, cell strain B was used for counter selection. The shift in signal between the blue and red peaks shows the level of aptamer binding.

As can be seen in Figure 1, by using a fluorescent stain, it is easy to see the presence of your target by seeing the shift in intensity.

 

Staining the cells

For Flow Cytometry there are multiple ways to stain cells. To do this you need to specifically attach a fluorophore to your cells of interest. There are several ways to do this but most methods use antibodies.

A benefit of using aptamers to assist with flow cytometry is that they are able to detect a multitude of targets. Aptamer binding is highly specific without the need for an immunogenic target. This helps when trying to identify unique biomarkers.

An advantage of using aptamers is that we design them to be optimised for the conditions you want to use them in. This intelligent design helps in 2 ways. The first is that if an aptamer has a high affinity to a target, it will lower the limit of detection. The second is that we can use less aptamer to detect the same number of cells. These two properties increase the functionality of aptamers for flow cytometry and reduce cost.

By incorporating a reactive linker onto the end of an aptamer different fluorophores can be added to suit your analytical needs. For example by using multiple aptamers in this way, different dyes can be conjugated to each aptamer, allowing you to distinguish closely related cells.

 

If you want to explore how aptamers can be used for your research, discovery and development projects then please contact us using the form below.