Refractive index to evaluate staining specificity of extracellular vesicles by flow cytometry

Abstract

Extracellular vesicles (EVs) in plasma are commonly identified by staining with antibodies and generic dyes, but the specificity of antibodies and dyes to stain EVs is often unknown. Previously, we showed that platelet-depleted platelet concentrate contains two populations of particles >200 nm, one population with a refractive index (RI) < 1.42 that included the majority of EVs, and a second population with an RI > 1.42, which was thought to include lipoproteins. In this study, we investigated whether EVs can be distinguished from lipoproteins by the RI and whether the RI can be used to determine the specificity of antibodies and generic dyes used to stain plasma EVs. EVs and lipoproteins present in platelet-depleted platelet concentrate were separated by density gradient centrifugation. The density fractions were analyzed by Western blot and transmission electron microscopy, the RI of particles was determined by Flow-SR. The RI was used to evaluate the staining specificity of an antibody against platelet glycoprotein IIIa (CD61) and the commonly used generic dyes calcein AM, calcein violet, di-8-ANEPPS, and lactadherin in plasma. After density gradient centrifugation, EV-enriched fractions (1.12 to 1.07 g/mL) contained the highest concentration of particles with an RI < 1.42, and the lipoproteinenriched fractions (1.04 to 1.03 g/mL) contained the highest concentration of particles with an RI > 1.42. Application of the RI showed that CD61-APC had the highest staining specificity for EVs, followed by lactadherin and calcein violet. Di-8-ANEPPS stained mainly lipoproteins and calcein AM stained neither lipoproteins nor EVs. Taken together, the RI can be used to distinguish EVs and lipoproteins, and thus allows evaluation of the specificity of antibodies and generic dyes to stain EVs.

Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?

“…Calcein staining was dim to moderate, yet the distinction between positive and negative events, remained discernible by visual inspection ( Figure 1 , left panel). These findings seem at odds with those of De Rond et al [ 15 ] that deemed Calcein as scarcely sensitive. We can only conjecture as to why our findings differ from previous results because the lack of a detailed description of the gating strategy and particle size in the De Rond study [ 15 ] make a comparison difficult.…”

Ticagrelor attenuates the increase of extracellular vesicle concentrations in plasma after acute myocardial infarction compared to clopidogrel

“…Flow cytometry (A60‐Micro, Apogee Flow Systems) was used to determine the concentration of EV subtypes in platelet‐depleted plasma. The reported concentrations describe the number of particles (a) that exceeded the side scatter threshold, corresponding to a side scattering cross section of 10 nm 2 , (b) with a diameter >200 nm as determined by Flow‐SR, (c) having a refractive index <1.42 to omit false positively labeled chylomicrons, and (d) that are positive at the fluorescence detector(s) corresponding to the used label(s), per mL of platelet‐depleted plasma. We aimed to label activated platelets (CD61 + /P‐selectin + ), fibrinogen + , leukocytes (CD45 + ), ECs (CD31 + /CD146 + ), erythrocytes (CD235a + ), and all procoagulant EVs (PS + ) in platelet‐depleted plasma.…”

Towards defining reference materials for measuring extracellular vesicle refractive index, epitope abundance, size and concentration

“…material if it is intended for use with optical analysis techniques. Current literature suggests that while the refractive index of EVs is lower than reference materials such as silica, it is variable, Figure 5 [22,23,34,40]. Indeed, the effective refractive index of EVs will never be a single number due to an EV being a core-shell model, where the ratio of the shell (membrane) to the core (cytosolic portion) increases as EV size decreases.…”