High‐Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies

Abstract

Extracellular vesicles (EVs) are released from basically all cells. Over the last decade, small EVs (sEVs; 50–150 nm) have gained enormous attention in diagnostics and therapy. However, methodological limitations coupled to the lack of EV standards leave many questions in this quickly evolving field unresolved. Recently, by using enhanced green fluorescent protein (eGFP)‐labeled sEVs as biological reference material, we systematically optimized imaging flow cytometry for single sEV analysis. Furthermore, we showed that sEVs stained with different fluorescent antibodies can be analyzed in a multiparametric manner. However, many parameters potentially affecting the sEV staining procedure still require further evaluation and optimization. Here, we present a concise, systematic evaluation of the impact of the incubation temperature (4°C, room temperature and 37°C) during sEV antibody staining on the outcome of experiments involving the staining of EVs with fluorescence‐conjugated antibodies. We provide evidence that both the staining intensity and the sample recovery can vary depending on the incubation temperature applied, and that observed differences are less pronounced following prolonged incubation times. In addition, this study can serve as an application‐specific example of parameter evaluation in EV flow cytometry. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Characterization of Extracellular Vesicles Labelled with a Lipophilic Dye Using Fluorescence Nanoparticle Tracking Analysis

“…This indicates that the labeling ratios for fl-NPs of EVs were less affected by different incubation temperatures. Although, in another investigation, using high-resolution flow cytometry, Tertel et al studied the impact of incubation temperature on labeling of enhanced GFP EVs with different fluorescence-conjugated antibodies [42]. This observation and our study showed generally higher labeling efficiency of EVs at the highest incubation temperature, though the detection methods employed were different.…”

Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques

“…IFCM was performed on the AMNIS ImageStreamX Mark II Flow Cytometer (AMNIS/Luminex, Seattle, WA, USA) as described before (26,28). Details for all antibodies used are provided in Suppl.…”

“…Recently, we set up protocols to label sEVs with fluorescent conjugated anti-CD9, anti-CD63 and anti-CD81 antibodies in otherwise non-processed cell culture supernatants, allowing us to immediately analyze the labeled EVs by IFCM (26,28). Being interested in uEVs in the context of biomarker research, we explored whether a comparable labeling technology could be used for the detection of uEVs in fresh void urine samples of healthy donors (Fig.…”

“…Currently, the validity of EV preparation methods is frequently judged by particle quantification technologies, e.g., by nanoparticle tracking analysis (NTA), which we and others introduced in 2011 as an "exosome" quantification method (24,25). However, upon comparing NTA with imaging flow cytometry (IFCM) analysis, the latter allowing single EV analyses even in nonprocessed EV containing samples (26)(27)(28), we had to learn that depending on the initial sample material and the applied preparation method, obtained EV samples contain far more particles than EVs. Indeed, protein aggregates that are formed in urine appear in NTA as small particles which are indistinguishable from uEVs (29).…”

Aiming to Compare Apples to Apples: Analysis of Extracellular Vesicles and Other Nanosized Particles by Flow Cytometry

“…However, because small EVs bear few antigens, EV immunofluorescence requires careful optimization and calibration of a sort not commonly encountered in the published literature. Tertel and colleagues (page 602‐609) provide a clear example of the optimization and reporting of EV immunofluorescence as applied to an imaging flow cytometer (12). By evaluating the key staining parameters of concentration, temperature, and time, and performing appropriate calibration to report fluorescence in absolute units of MESF (molecules of equivalent soluble fluorochrome), they demonstrate how to produce quantitative and reproducible EV immunofluorescence data.…”