Marker identification and quantification by StrataQuest, and confocal analysis of EV-containing tissue sections. Marker identification and quantification.

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Marker identification and quantification by StrataQuest, and confocal analysis of EV-containing tissue sections. Marker identification and quantification by StrataQuest, and confocal analysis of EV-containing tissue sections. (A) StrataQuest analysis algorithm. First, nuclei are identified and quantified (gated) through propidium iodide (PI) assessment (gated nuclei: 86.53%; first two images and gate 1). Around the nuclei, the software calculates and demarcates a small area that represents the cell cytoplasm and boundary (green net-like structure in image 2). Subsequently, a marker image is superimposed, demarcating the area of interest (here area of EV deposition in a lymph node, images 3 and 4, gate 2), which is set to 100% in order to calculate cell sub-populations in this area. Subsequently, an additional marker is superimposed and gated on the previous gate (43.22% of cells in the EV area, gate 3). This calculation can be performed for different individual markers (e.g., Fig 4), or continued for assessing multiple marker co-expression in one cell (e.g., Table 1). Scale bars represent 100 μm. (B) Analysis of cellular EV uptake in tissue. A tissue section as described in Fig 4B (maDC-EV 6 h after injection) was analyzed by confocal microscopy in order to demonstrate that EV were ingested by cells and not merely deposited by injection. To indicate the cytoplasm, the cells were stained for CD11b. EV are indicated by red color and nuclei by DAPI. Scale bars represent 25 μm. Stefan Schierer et al. LSA 2018;1:e201800093 © 2018 Schierer et al.