Fig. 1.

a,c Flow-sorted FXTAS inclusions exhibit the same size and autofluorescent properties as in situ FXTAS inclusions. FXTAS inclusions exhibit strong autofluorescence at 480 nm and 545 nm, and weak autofluorescence at 360 nm and 620 nm wavelengths. Inclusions sorted by flow cytometry were verified by microscopy to confirm that sorted inclusions exhibit no significant difference from FXTAS inclusions viewed in situ. Slides were stained with DAPI only and viewed at 100x. Orange arrows denote inclusions, upper left labels indicate the wavelength used for the image, and upper right labels indicate the exposure level used to take the image. No postprocessing adjustments were made to brightness/contrast. b Fractions enriched in inclusions and submitted for FACS contain a population of FXTAS-specific particles identified by size and fluorescence properties. Logarithmic scaling was used on the detectors assigned to laser light scatter measurements (Inclusion scatter), and larger aggregates were removed by plotting the duration of 90° laser light scatter to remove objects with markedly increased laser dwell rates relative to the shorter transit times of single particles (Single inclusions). Sorted particles were identified as a population in FXTAS samples that was absent in control samples which exhibited strong green fluorescence emission and weak red fluorescence emission (gates in Green autofluorescence and Red autofluorescence, respectively). d Inclusions sorted sequentially by nuclear isolation, sucrose gradient, and flow cytometry are of high concentration and purity. Sorted inclusion samples viewed at 60x show high purity, with 80-90% of autofluorescent particles displaying autofluorescent properties consistent with FXTAS inclusions. 10-20% of particles (orange arrows) display a high level of autofluorescence in the far-red wavelengths, indicating that a small degree of non-inclusion debris may be present. Scale bars = 5 μm