Figure 2.

Exosomal RNA cargo is delivered to macrophages. Exosomes were purified from 400–800 mL supernatant of L. donovani axenic amastigotes, protein concentration was determined by Micro BCA, and exosomes were stained with a green fluorescent RNA-specific dye. PMA-differentiated THP-1 cells were incubated for 2 hours with labelled exosomes at either 37°C or 4°C to inhibit phagocytosis. A. Nanosight size profile of purified exosomes, B. Cells were treated with different concentrations of labelled exosomes as indicated and analysed by flow cytometry. Histograms were drawn, median fluorescence intensity (MFI) of cells was recorded, and the mean of 3 independent experiments was calculated (error bars represent standard error of the mean, SEM). C. Confocal microscopy of cells incubated with 10 μg/mL exosomes (green) at 4°C (left) or 37°C (right). Cells were stained with phalloidin-Alexa 594 to detect actin (red) and DAPI to detect nuclei (blue). Confocal microscopy was done with a Leica DMIRE2 inverted microscope equipped with a SP2 AOBS laser scanning head. Images were taken with a 63× magnification objective. Images are representative of 3 independent experiments.