Flow cytometry with aptamers

Flow cytometry is a technique used to identify biomarkers on cell surfaces and intracellular molecules. Flow cytometers measure fluorescently-labelled affinity reagents, like antibodies, that are bound to specific cell markers to differentiate between cells expressing these markers.

Aptamers for flow cytometry are allowing researchers to pursue biomarkers that have proven intractable with antibodies, and identify new biomarkers through cell-based discovery processes, supporting a range of fields from cancer diagnostics to food and drink analysis and stem cell research.

How does flow cytometry work?

It starts with your cells. Suspend your culture in buffer, label it with a fluorescently-tagged affinity reagent specific to the biomarker you’re interested in, whether aptamer or antibody, then wash away anything that didn’t bind. This step matters as it ensures the fluorescence you measure in flow cytometry comes from your bound aptamer, not background signal drifting around in the sample.

The labelled cells then flow into the cytometer, where hydrodynamic focusing lines them up single file so each one passes the laser individually. As they do, two readouts are captured at once:

1. Light scatter: forward scatter reflects cell size, while side scatter reflects granularity, giving a quick physical profile of each cell.
2. Fluorescence: As a labelled cell crosses the laser, its fluorophore is excited and emits light, which the detectors pick up. Across thousands of cells, this builds a picture of both how strongly each cell expresses the biomarker and what proportion of the population expresses it at all.

The results then give you a detailed, cell-by-cell view of biomarker expression across your whole sample, in minutes.

You can also take this further with multiplexing by labelling cells for several biomarkers at once, using aptamers conjugated to fluorophores with distinct excitation and emission spectra. Each fluorophore can be distinguished from the others by its relevant light absorption and emission, meaning multiple lasers can profile the cells at once to give corresponding signals from each aptamer label on the cell simultaneously. This provides the expression data for several markers in parallel on each cell to give a more complete picture of the cells protein expression profile. This is widely used in stem cell phenotyping, where distinguishing cell populations often depends on looking at more than one marker together and including both positively and negatively expressed marker profiles to identify specific cell sub-populations.

The benefits of aptamers for flow cytometry

Aptamers have been used as probes in flow cytometry for many decades, and offer lots of advantages over protein-based alternatives, like antibodies.

Aptamers are based on a known sequence and are made by chemical synthesis. This is a highly consistent manufacturing method, meaning aptamers are have the highest lot-to-lot consistency, overcoming reproducibility issues that have plagued life science research.

They can be functionalised with a range of fluorophores to create panels of aptamers or combination panels of aptamers and antibodies for multiplexing.

Aptamers are more thermostable than antibodies, allowing them to be shipped at room temperature sparing researchers the hassle of freezers and ice packs, and making day-to-day handling that bit simpler.

A comparison of assay costs of custom synthesised RNA aptamers and commerical antibodies showed as little costs as US$0.002 for aptamer reagents versus US$2 for antibody reagents.

As part of our discovery process for flow cytometry reagents, we use whole cells as targets, either alone or combined with specific protein biomarkers. Using cells within the discovery process, means that the aptamers we generate recognise biomarkers in their natural, cell-expressed form, not just as isolated proteins. We also use flow cytometry within our cell discovery process, meaning every aptamer developed for flow cytometry is validated for performance as part of that same process.

FACS-based selection of flow cytometry optimised Optimer reagents.

A benefit of using aptamers for flow cytometry is that they can often support where high quality antibodies or alternatives might be missing. Aptamers can be developed to be highly target-specific, and because they are developed through in vitro processes they can be generated to non-immunogenic targets to help identify unique biomarkers or increase specificity in a targeted way.

Applications of aptamers in flow cytometry

Aptamers have been successfully used in flow cytometry for a wide variety of applications, from labelling of cell surface markers to cancer cell detection.

Because flow cytometry lends itself to so many different uses, it’s become a go-to technique for researchers across a wide range of fields, including drug testing, industrial bioprocesses, cell line development, stem cell research, and diagnostics.

A whole-cell discovery approach for aptamers

Our whole-cell discovery approach lets us identify aptamers for any two cell types that differ in their surface expression, such as malignant versus healthy cells or subpopulations within a single disease-related cell type. This can support identification of new disease-state biomarkers, as well as pinpointing the optimal biomarker for a specific application, such as targeted drug delivery into a cell. Using this approach, aptamers are only selected once they’ve demonstrated internalisation, so researchers avoid chasing mechanistic dead ends further down the line.

Prior knowledge of the target biomarker isn’t needed to get started. Aptamer discovery generates specific binders to the cell surface first, and biomarker identification can then be carried out downstream using mass spectrometry. This flexibility means the approach can deliver a single aptamer for a single biomarker, multiple aptamers against one biomarker to boost sensitivity (similar to a polyclonal antibody mix), or in cases like detecting multiple strains of a single species, aptamers targeting several different biomarkers in parallel.

If you want to explore how aptamers can be used for your research, discovery and development projects then get in touch.

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