High-resolution imaging flow cytometry reveals impact of incubation temperature on labeling of extracellular vesicles with antibodies

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, researchers from the Karolinska Institutet and the University of Duisburg‐Essen systematically optimized imaging flow cytometry for single sEV analysis. Furthermore, they 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, the researchers 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. They 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.

IFCM facilitates robust analysis of fluorescent sEVs

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(A) Gating strategy applied to identify SSClow eGFP+ sEV events [eGFP+] with X‐axis showing FITC MESF calibrated data. (B) Evaluation of reproducibility of IFCM analyses in 96‐well plates. Precleared and filtrated identical, unstained CM samples derived from THP‐1:CD63eGFP cells were pipetted into a 96‐well plate and measured with the built‐in autosampler in three independent experiments to compare, respectively, obtained concentration (left graph) and fluorescence intensity. Cutoffs for gating on SSC(low) eGFP(+) events were chosen based on background events in PBS only controls for eGFP (>40 FITC MESF) and based on previous characterization of eGFP‐tagged sEVs and to include the main eGFP(+) population for SSC (<1,000 a.u.). Coefficients of variation (cVs) were calculated dependent on which rows of wells were measured. 
Tertel T, Bremer M, Maire C, Lamszus K, Peine S, Jawad R, Andaloussi SEL, Giebel B, Ricklefs FL, Görgens A. (2020) High-Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies. Cytometry A [Epub ahead of print]. [article]

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