Nanoparticles for Flow Cytometry

Typical flow cytometry dot plot with SSC on y-axis and fluorescence due to cell staining with Mouse anti Human CD8 NTB660 on x-axis

Application: Flow Cytometry

Conventional flow cytometry can be described, in a simplistic way, as a technique which allows small entities to be moved in a faster-moving sheath fluid to encounter the beam of a light source. In this process, a sample containing cells or particles is injected into the instrument and then focused on flow one cell at a time through a laser beam. The light scattered is characteristic of the cells and their components. When cells are marked with fluorescent probes, light is absorbed and then emitted in a characteristic wavelength collected by specific detectors. Not long ago, different types of cytometry were developed and are now on the crest of the wave. This is the case of spectral flow cytometry in which the evaluation is due to the whole spectrum from visible to near-infrared region and not to the signal’s peak.

State of the art

Recent years brought a breath of freshness because of the constant, and increasing, needs for the analysis of multiple parameters. After the expansion of lasers and detectors, it is now the time for the growth in the number of available fluorochromes. Tandem dyes were developed to increase the fluorescent parameters of each experimental session. They are composed of two linked fluorophores: one acts as the donor and the other as the acceptor. The donor fluorophore is excited by the flow cytometer laser and transmits its absorbed energy to the acceptor one, which then emits fluorescence signal exploiting the Forster Resonance Energy Transfer (FRET). Unfortunately, tandem dyes may easily and irreversibly decouple or degrade after exposure to light, temperature variation, or fixation, resulting in the emission in the donor fluorophore's spectrum. An additional drawback of fluorescent conjugates is the low number of fluorescent molecules per antibody: degree of labeling (DOL). In the case of small fluorescent molecules, it ranges from 4 to 9, but in the case of bigger fluorescent molecules it is, on average, 1 fluorescent molecule per antibody.

AcZon’s solution

AcZon brought nanotechnology into the field of flow cytometry confining these highly sensitive dyes in the nanoparticle silica matrix. This protective shield avoids the above-mentioned unpleasant effects offering high reagent stability. NanoChromes and NanoTandems, thanks to the concentrative effect due to the employment of the spheric nanotool, significantly enhance the DOL with a consequent increase in the signal intensity assuring an improved discrimination between negative and positive populations.