Biology subjects

Arkesteijn, G. J. A.

Publications and source records attributed to Arkesteijn, G. J. A..

4 recordsLinked to original sources

bioRxiv2026

Characterization of nanoparticles and fluorescent recombinant extracellular vesicles with three high-sensitivity flow cytometers

High-sensitivity flow cytometry (FC) allows multiparametric analysis of nanoparticles (NPs) and extracellular vesicles (EVs). With new instruments available, studies that evaluate their performance using the same materials in a controlled environment are required. Here, we performed a comparative study to investigate the capabilities of three flow cytometers, namely the NanoFCM (NF), BD Influx (IF) and CytoFLEX LX (CF). Firstly, we analyzed a mixed population of silica NPs (SiNPs, 68, 91, 114 and 155 nm) by using light-scatter based detection thresholds (SSC, FSC, VSSC) across a concentration range from 106 to 109 particles/mL. Next, we analyzed fluorescent recombinant EVs (rEVs) by comparing light-scatter based thresholding (488 nm SSC available for all platforms), the combination of SSC thresholding with a fluorescent gate, and fluorescent thresholding for their qualitative and quantitative analysis. We here provide the strengths and limitations for each platform regarding the analysis of differently sized NPs at different sample concentrations.

cell biology↗

bioRxiv2023

Maternal allergic sensitization affects the T cell modulatory capacity and molecular cargo of milk derived extracellular vesicles

BackgroundHuman milk extracellular vesicles (EVs) affect various cell types in the gastrointestinal tract, including T cells, and play a role in the development of the newborns immune system by delivering specific molecular cargo to target cells. Although maternal allergic sensitization alters the composition of milk, it is unknown whether this impacts the function of milk EVs. Therefore, we analyzed the T cell modulatory capacity and compared the protein and miRNA cargoes of EVs from milk of allergic and non-allergic mothers. MethodsEVs were isolated from human milk from allergic and non-allergic donors by differential centrifugation, density gradient floatation and size exclusion chromatography. Functional modulation of primary human CD4+ T cells by EVs was assessed in vitro. Proteomic analysis and small RNA sequencing was performed on milk EVs to evaluate protein and miRNA abundance and to identify cellular targets of this EV cargo in relevant T cell signaling pathways. ResultsT cell proliferation, activation and cytokine production were suppressed in the presence of milk EVs. Remarkably, milk EVs from allergic mothers inhibited T cell activation to a lesser extent than EVs from non-allergic mothers. Integrative multi-omics analysis identified EV cargo of which the cellular targets could be linked to differential modulation of T cell activation-associated processes . ConclusionsMilk EVs from non-allergic mothers are stronger inhibitors of T cell activation compared to milk EVs from allergic mothers. This altered functionality might be linked to changes in miRNA and protein cargo that modulate T cell signaling pathways in an integrative manner.

immunology↗

bioRxiv2022

Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact single EV-based analysis and biomarker profiling, we investigated whether under physiological conditions LPPs and EVs can associate by using cryo-electron tomography, label-free synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high resolution single particle flow cytometric analysis. Furthermore, we evaluated the impact on flow cytometric analysis in the absence or presence of different types of LPPs using in vitro spike-in experiments of purified tumor cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrated that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations can influence EV analysis in a quantitative and qualitative manner. Our findings demonstrate that the biological colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis.

cell biology↗

bioRxiv2021

Considerations for MESF-bead based assignment of absolute fluorescence values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry is commonly used to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units to indicate fluorescence intensity. This hampers interlaboratory and inter-platform comparisons. We investigated the use of molecules of equivalent soluble fluorophores (MESF)-beads for assignment of fluorescence values to NPs and EVs by comparing two FITC-MESF bead sets as calibrators on different flow cytometry platforms (BD Influx, CytoFLEX LX and SORP BD FACSCelesta). Next, fluorescence signals of NPs and EVs were calibrated using different sets of FITC and PE-MESF beads. Fluorescence calibration using beads designed for cellular flow cytometry allowed inter-platform comparison. However, the intrinsic uncertainty in the fluorescence assignment to these MESF beads impacts the reliable assignment of MESF values to NPs and EVs based on extrapolation into the dim fluorescence range. Our findings demonstrate that the use of the same set of calibration materials (vendor and lot number) and the same number of calibration points, greatly improves robust interlaboratory and inter-platform comparison of fluorescent submicron sized particles.

biophysics↗