Unique Calibrators Derived from Fluorescence‐Activated Nanoparticle Sorting for Flow Cytometric Size Estimation of Artificial Vesicles: Possibilities and Limitations

Abstract

The use of high-throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer-sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence-activated cell sorting (FACS), here coined fluorescence-activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore-labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS-sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS-sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS-sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS-calibrators against standard methods for sizing nanoparticles, including NTA and cryo-transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30-300 nm) artificial vesicles.TO be able to analyze multiple parameters of individual nanoparticles, including their size, in a high-throughput manner has long been a goal in nanoparticle research. Traditional tools for nanoparticle characterization are often limited by a low throughput, such as microscopy-based methods, or restricted to single-property measurements using ensemble-based methods. The potential problem with employing bulk absorption and emission spectroscopy-based methods or dynamic light small-angle X-ray or neutron scattering techniques is that the ensemble average information they provide

Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis

“…We applied purified PICK1 to small unilamellar liposomes (SUVs), prepared from bovine brain extract (Folch fraction 1, ~2.5 µg/mL) and labeled with the lipophilic dye (DiD) followed by incubation for 1 hour (h). The distribution of fluorescence intensities of single events, representing individual liposome, was plotted using kernel density estimates (Figure 7B, grey line) and the distribution matched previously reported distributions, with the peak at log 2.4 corresponding to liposome diameters between 50 and 100 nm (35). Upon incubation with PICK1, a small but reproducible shift in the distribution towards lower intensities was observed (Figure 7B).…”

Technologies and Standardization in Research on Extracellular Vesicles

“…Standard alternatives to silica and polystyrene beads were also introduced for EV FCM analysis. For example, liposomes or hollow organosilica beads scatter light similarly to EVs [137,138]. Also, differences in the hardware from different manufacturers can cause variabilities, which should be taken into account when comparing data from different flow cytometers [131].…”

The effect of ionising radiation on the phenotype of bone marrow-derived extracellular vesicles

“…First, we used a commercially available calibration bead mixture called Gigamix, which contains beads with different well-defined diameters ranging between 100 nm and 900 nm and inserted the gate in a way to identify particles ranging between 100 nm and 500 nm. This method is generally accepted, 28,29 although recently its accuracy has been questioned, 1,30 since synthetic solid beads and biological vesicles have different refractive indices (e.g., 200 nm EVs scatter 40-300-fold less light than 200 nm polystyrene beads at a wavelength of 405 nm) for which reason polystyrene beads cannot be used directly for the sizing of EVs. 31,32 Chandler et al demonstrated that 400 nm polystyrene beads likely correlate with 1,000 nm microparticles.…”