Biology subjects

Bohn, A. B.

Publications and source records attributed to Bohn, A. B..

4 recordsLinked to original sources

bioRxiv2025

Dedicated nanoparticle flow cytometry for single extracellular vesicle phenotyping: Performance of the CytoFLEX Nano

Accurate discrimination of extracellular vesicles (EVs) from non-vesicular nanoparticles and robust phenotyping of individual EVs directly within complex biofluids are essential to advance understanding of EV biology and to realize their potential as biomarkers and therapeutic agents. Conventional flow cytometers, originally designed for cellular analysis, lack the scatter and fluorescence sensitivity, dynamic range, and event-rate control required for quantitative characterization of nanoscale vesicles and are particularly susceptible to coincident (swarm) detection. Dedicated nanoparticle flow cytometers have been developed to address these limitations, and here we systematically evaluate the suitability of the CytoFLEX Nano (Beckman Coulter) for EV analysis. Using a series of calibration beads of different materials, fluorescent liposomes, and EVs isolated from peritoneal fluid and cell culture medium, we assess size-detection thresholds, scatter and fluorescence sensitivity, dynamic range, and volumetric counting accuracy. These data provide practical guidance for implementing nanoscale flow cytometry in EV research and support the informed adoption of dedicated nanoparticle cytometers in studies adhering to current EV reporting standards.

cell biology↗

bioRxiv2025

Inter-Instrument Quantification of Fluorescence Read-out Signal Using DNA Origami Calibrator Beads

Comparing experimental results across different instruments and laboratories is challenging due to variations in instrument sensitivity and resolution, particularly for analyzing nano-sized and dimly fluorescing particles like viruses and extracellular vesicles. To address this challenge, we conducted a study using fluorescent DNA calibrator beads to test, calibrate, and compare the sensitivity of five different flow cytometers. Our approach involves DNA beads produced through a bottom-up DNA origami method. These beads contain a defined number of fluorophores (0-220) and exhibit no autofluorescence. Fluorophores are precisely conjugated onto the beads in a controlled array, minimizing dye interactions. This allows triggering on one fluorophore and calibration based on another, facilitating precise and accurate calibration in absolute fluorophore numbers. We tested five flow cytometers--FACSAria III, NovoCyte 3000YBG, NovoCyte Quanteon 4500, LSRFortessa, and CytoFlex S--using Cy5 and FAM DNA calibrator beads. We investigated various instrument parameters, including count rate, trigger and calibration gain, gain amplification linearity, and fluorescence trigger sensitivity. Using fluorescent triggering, we demonstrated the capability to detect dimly fluorescent particles, enabling instrument calibration and fluorescence sensitivity comparison using DNA calibrators. In conclusion, our study offers a robust method for addressing instrument fluorescence channel resolution in absolute fluorophore numbers in the analysis of nano-sized and dimly fluorescent particles.

biophysics↗

bioRxiv2024

The limited capacity of bioaerosols to serve as cloud-condensation nuclei may restrict their potential to initiate ice formation in mixed-phase clouds

Bioaerosols are gaining prominence due to their ability to nucleate ice in clouds at high subzero temperatures, thereby impacting cloud characteristics, and longevity. Acting first as cloud condensation nuclei (CCN) and second as ice nucleating particles, bioaerosols induce ice formation through immersion freezing. Nevertheless, insights into bioaerosol ability to act as CCN in situ are currently lacking. We simultaneously collected bioaerosols from the condensed and interstitial phase of clouds using size selective inlets during autumn 2020 from the Otlica observatory (Slovenia). With a polarization Raman Lidar, we confirmed the coexistence of ice particles and liquid droplets during one of the three sampling campaigns. Using spectral flow cytometers, the PINGUIN cold-stage setup and MiSeq-facilitated amplicon sequencing to assess bacterial and fungal communities, we found that both bioaerosols and biogenic ice-nucleating particles were significantly more abundant in the interstitial phase compared to the condensed phase, suggesting that they were poor CCN, unable to compete with more hygroscopic particles. Most taxa did not exhibit preferential partitioning, suggesting that surface properties generally did not significantly influence their behaviour as CCN. Our results underline the need to systematically investigate the relation between hygroscopicity and ice-nucleating activity of bioaerosols to understand their in-situ effects on cloud formation. SynopsisThe relationship between the ability of bioaerosols to nucleate cloud droplets and ice particles has not been determined. This study found that bioaerosols are poor cloud condensation nuclei which may impair their ability to nucleate ice and affect cloud formation and climate.

microbiology↗

bioRxiv2023

Analysis of cortical cell polarity by imaging flow cytometry

Metastasis is the main cause of cancer-related death and therapies specifically targeting metastasis are highly needed. Cortical cell polarity (CCP) is a pro-metastatic property of circulating tumor cells (CTCs) affecting their ability to exit blood vessels and form new metastases that constitutes a promising point of attack to prevent metastasis. However, conventional fluorescence microscopy on single cells and manual quantification of CCP are time-consuming and unsuitable for screening of regulators. In this study, we developed an imaging flow cytometry (IFC)-based method for high-throughput screening of factors affecting CCP in melanoma cells. The artificial intelligence (AI)-supported analysis method we developed is highly reproducible, accurate, and orders of magnitude faster than manual quantification. Additionally, this method is flexible and can be adapted to include additional cellular parameters. In a small-scale pilot experiment using polarity-, cytoskeleton-or membrane-affecting drugs, we demonstrate that our workflow provides a straightforward and efficient approach for screening factors affecting CCP in cells in suspension and provide insights into the specific function of these drugs in this cellular system. The method and workflow presented here will facilitate large-scale studies to reveal novel cell-intrinsic as well as systemic factors controlling CCP during metastasis.

cell biology↗