Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation

Essentials Platelet extracellular vesicles (EVs) concentrations measured by flow cytometers are incomparable. A model is applied to convert ambiguous scatter units to EV diameter in nanometer. Most included flow cytometers lack the sensitivity to detect EVs of 600 nm and smaller. The model outperforms polystyrene beads for comparability of platelet EV concentrations. Summary: Background Detection of extracellular vesicles (EVs) by flow cytometry has poor interlaboratory comparability, owing to differences in flow cytometer (FCM) sensitivity. Previous workshops distributed polystyrene beads to set a scatter-based diameter gate in order to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insights into the diameter of detected EVs. Objectives To evaluate gates based on the estimated diameter of EVs instead of beads. Methods A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates in order to measure the concentration of CD61–phycoerythrin-positive platelet EVs. Results Of the 46 evaluated FCMs, 21 FCMs detected the 600–1200-nm EV diameter gate. The 1200–3000-nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration interlaboratory variability of 81% as compared with 139% with the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL min−1 differed six-fold in measured flow rates between instruments. Conclusions EV diameter gates improve the interlaboratory variability as compared with previous approaches. Of the evaluated FCMs, 24% could not detect 400-nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity

Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation

BACKGROUND Detection of extracellular vesicles (EVs) by flow cytometry (FCM) has poor inter-laboratory comparability due to differences in FCM sensitivity. Previous workshops distributed polystyrene beads to set a scatter-based diameter gate to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insight into the diameter of detected EVs. OBJECTIVES To evaluate gates based on the estimated diameter of EVs instead of beads. METHODS A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates to measure the concentration of CD61-phycoerythrin positive platelet EVs. RESULTS Of the 46 evaluated FCMs, 21 FCMs detected the 600-1,200 nm EV diameter gate. The 1,200-3,000 nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration inter-laboratory variability of 81% compared to 139% by the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL/minute differed 6-fold in measured flow rates between instruments. CONCLUSIONS EV diameter gates improve the inter-laboratory variability compared to previous approaches. Of the evaluated FCMs, 24% could not detect 400 nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity. This article is protected by copyright. All rights reserved

Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation

BACKGROUND Detection of extracellular vesicles (EVs) by flow cytometry (FCM) has poor inter-laboratory comparability due to differences in FCM sensitivity. Previous workshops distributed polystyrene beads to set a scatter-based diameter gate to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insight into the diameter of detected EVs. OBJECTIVES To evaluate gates based on the estimated diameter of EVs instead of beads. METHODS A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates to measure the concentration of CD61-phycoerythrin positive platelet EVs. RESULTS Of the 46 evaluated FCMs, 21 FCMs detected the 600-1,200 nm EV diameter gate. The 1,200-3,000 nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration inter-laboratory variability of 81% compared to 139% by the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL/minute differed 6-fold in measured flow rates between instruments. CONCLUSIONS EV diameter gates improve the inter-laboratory variability compared to previous approaches. Of the evaluated FCMs, 24% could not detect 400 nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity. This article is protected by copyright. All rights reserved

Role of soil rhizobacteria in phytoremediation of heavy metal contaminated soils

Heavy metal pollution of soil is a significant environmental problem and has its negative impact on human health and agriculture. Rhizosphere, as an important interface of soil and plant, plays a significant role in phytoremediation of contaminated soil by heavy metals, in which, microbial populations are known to affect heavy metal mobility and availability to the plant through release of chelating agents, acidification, phosphate solubilization and redox changes, and therefore, have potential to enhance phytoremediation processes. Phytoremediation strategies with appropriate heavy metal-adapted rhizobacteria have received more and more attention. This article paper reviews some recent advances in effect and significance of rhizobacteria in phytoremediation of heavy metal contaminated soils. There is also a need to improve our understanding of the mechanisms involved in the transfer and mobilization of heavy metals by rhizobacteria and to conduct research on the selection of microbial isolates from rhizosphere of plants growing on heavy metal contaminated soils for specific restoration programmes