Lipid-based strategies used to identify extracellular vesicles in flow cytometry can be confounded by lipoproteins: Evaluations of annexin V, lactadherin, and detergent lysis

Abstract

Flow cytometry (FCM) is a popular method used in characterisation of extracellular vesicles (EVs). Circulating EVs are often identified by FCM by exploiting the lipid nature of EVs by staining with Annexin V (Anx5) or lactadherin against the membrane phospholipid phosphatidylserine (PS) and evaluating the specificity of the labels by detergent lysis of EVs. Here, we investigate whether PS labelling and detergent lysis approaches are confounded by lipoproteins, another family of lipid-based nanoparticles found in blood, in both frozen and fresh blood plasma. We demonstrated that Anx5 and lactadherin in addition to EVs stained ApoB-containing lipoproteins, identified by the use of fluorophore-labelled polyclonal ApoB-antibody, and that Anx5 had a significantly larger tendency for labelling lipoprotein-bound PS than lactadherin. Furthermore, detergent lysis resulted in a decrease in both EV and lipoprotein events and especially lipoproteins positive for either Anx5 or lactadherin. Taken together, our findings pose concerns to the use of lipid-based strategies in identifying EVs by FCM and support the use of transmembrane proteins such as tetraspannins to distinguish EVs from lipoproteins.

Keywords: VLDL; annexin V; chylomicrons; detergent lysis; exosomes; extracellular vesicles; flow cytometry; lactadherin; lipids; lipoproteins; phosphatidylserine; triton X-100.

FIGURE 1

Staining of ApoB‐containing lipoproteins with PE‐conjugated polyclonal anti‐ApoB‐48/100 antibody. (a) Scatter plots depicting large‐angle light scatter versus ApoB‐PE fluorescence of commercial VLDL pre‐diluted 8‐fold and stained with fluorescent anti‐ApoB antibody compared to matched isotype, unstained and buffer with reagent controls. (b) Scatter plots depicting large‐angle light scatter versus ApoB‐PE fluorescence of commercial chylomicrons pre‐diluted 4‐fold and stained with fluorescent anti‐ApoB antibody compared to isotype and unstained controls. (c) Scatter plots depicting large‐angle light scatter versus ApoB‐PE fluorescence of platelet poor plasma (PPP) pre‐diluted 32‐fold and stained with fluorescent anti‐ApoB antibody compared to isotype and unstained controls. Light scatter and fluorescence intensities in (A) and (B) are denoted in arbitrary units. (d) Serial dilution control of stained PPP presented with a linear relationship between ApoB event concentration not corrected for dilution factor (left y‐axis, black) and reciprocal dilution factor (x‐axis), while the median ApoB‐fluorescence for ApoB‐positive particles (right y‐axis, orange) was stable. Triangles depict the dilution factor used for all subsequent experiments. (e) Light scatter distributions of ApoB‐positive events in commercial VLDL (red) and chylomicrons (blue) overlayed with silica nanospheres (black, dotted) with an assumed similar RI of 1.47 to lipoproteins. Silica nanospheres from left to right: 100 nm; 180 nm; 300 nm; 590 nm; 1300 nm. ApoB: Apolipoprotein B; Chylo: Chylomicrons; LALS: Large‐angle light scatter (side scatter); VLDL: Very‐low density lipoprotein; Ab: Antibody; PPP: Platelet‐poor plasma

FIGURE 2

Co‐staining of Anx5, lactadherin and CD41 with ApoB‐containing lipoproteins. (a and b) Scatter plots of Anx5‐Cy5, lactadherin‐FITC and CD41‐BV510 versus ApoB‐PE fluorescence depicting the degree to which markers co‐stain with ApoB in (a) a pool of PPP that was stored at −80°C after collection or (b) freshly collected PPP from one of three different individuals (representative data). Samples were either stained with Anx5 or lactadherin, as these markers both label PS. Bar plots depict the percentage of marker‐positive events that were also positive for ApoB in specifically stained samples (blue) versus isotype controls (red). Bar plots for fresh PPP depicts the mean and standard deviation (error bars) for three individuals. (c) Dot plots (large‐angle light scatter versus Anx5 or lactadherin fluorescence) of ApoB+ (red) and CD41+ (green) events demonstrate a significant overlap of these populations on these parameters. Light scatter and fluorescence intensities are denoted in arbitrary units. Anx5: Annexin V; ApoB: Apolipoprotein B; CD41: Cluster of differentiation 41/platelet membrane glycoprotein IIb‐IIIa complex; Lact: Lactadherin; LALS: Large‐angle light scatter (side scatter); PPP: Platelet‐poor plasma

FIGURE 3

Effect of detergent lysis with Triton X‐100 on ApoB‐containing lipoproteins co‐stained with Anx5, lactadherin and CD41. (a) Scatter plots of Anx5‐Cy5 and lactadherin‐FITC versus ApoB‐PE fluorescence of a representative specifically stained fresh PPP sample and its detergent lysis controls depicting the degree to which the different fluorescently labelled populations are affected by detergent lysis. Fluorescence intensities is denoted in arbitrary units. (b) Bar plots of the concentrations of different marker‐positive populations in specifically stained fresh PPP (blue) and detergent lysis controls (red). Bar plots for fresh PPP depict the mean and standard deviation (error bars) for three individuals. Anx5: Annexin V; ApoB: Apolipoprotein B; CD41: Cluster of differentiation 41/platelet membrane glycoprotein IIb‐IIIa complex; Lact: Lactadherin

FIGURE 4

Illustrations of the similarities and differences between the large ApoB‐containing lipoproteins (VLDL and chylomicrons) and EVs. The yellow alpha‐helical structures on the VLDL and chylomicron represent ApoB‐100 and ApoB‐48, respectively. The blue spheres on all particles refer to phospholipids while the red spheres represent PS phospholipids. The additional coloured components on the EV represent different types of membrane proteins such as tetraspannins (green). Cholesterol and other membrane components including other types of apolipoproteins on the lipoproteins have been left out for the sake of simplicity