Chapter 5 126 Tetraspanin double-positive). Detergent treatment was applied on each sample after initial acquisition to discriminate between vesicular and non-vesicular events (Figure 2A). First, concentrations of fluorescent objects before and after detergent treatment were compared for each time point. For CFSE single-positive objects we observed a ~69% / 72% / 76% reduction in concentration after detergent treatment at T60 / T180/ T360, respectively (Figure 2B). This implies that a large fraction (~31% / 28% / 24%) of these objects represent non-vesicular (background) objects as they had not been dissolved by the detergent treatment. Consequently, the CFSE singlepositive population was excluded from further analysis. Detergent treatment reduced Tetraspanin single-positives (Figure 2C) and CFSE and Tetraspanin double-positives (Figure 2D) with 97.7% ± 0.004% and 99.8% ± 0.0002% respectively (normalized mean ± standard deviation, average reduction over all time points). Background levels (concentrations obtained after detergent treatment) of the Tetraspanin single-positive population resided around ~E6 objects/mL whereas the level of CFSE and Tetraspanin double-positives were observed to be <E5 objects/mL. These background levels were comparable to the baseline perfusate (T0) samples before detergent treatment. Second, for Tetraspanin single-positive and CFSE and Tetraspanin double-positive populations we observed ~43 / 56 / 57 and ~507 / 572 / 471 – fold increases after 60 / 180 / 360 minutes of NMP compared to T0, respectively (comparison of means). Comparing the mean concentration of Tetraspanin single-positive events to the mean concentration of Tetraspanin and CFSE double-positive events revealed ~4 / 5 / 6 – fold differences at 60 / 180 / 360 minutes of NMP respectively – indicating that less CFSE-positive EVs were detected as NMP progressed. Taken together, these findings indicate that 1) kidneys release EVs during NMP and 2) different subpopulations (based on tetraspanin expression in combination with the absence/presence of CFSE) can be identified using IFCM.
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