Panel A shows fluorescence microscopy images of LysoTracker Red DND-99 staining in NTg, SORL1 knockout, and unstained cells with brightfield views and enlarged insets. Panel B shows thresholded fluorescence microscopy images highlighting LysoTracker-positive objects in NTg and SORL1 knockout cells. Panel C shows a bar graph quantifying the number of LysoTracker-positive objects per cell. Panel D shows a bar graph quantifying LysoTracker mean fluorescence intensity per object. Panel E shows a bar graph comparing LysoTracker-positive object surface area. Panel F shows a bar graph comparing LysoTracker-positive object volume. Panel G shows a bar graph comparing the distance from the object center to the cell surface. Panel H shows a histogram comparing LysoTracker fluorescence intensity in unstained, NTg, and SORL1 knockout cells. Panel I shows a bar graph comparing LysoTracker mean fluorescence intensity between NTg and SORL1 knockout cells.
SorLA does not play a role in lysosome maintenance in Ramos-Cas9 B cells. (A) Confocal micrographs showing NTg and SORL1KO Ramos-Cas9 B cell lines labelled with LysoTracker Red DND-99 (yellow). NTg (left), SORL1KO (center), and unstained cells (right); inset (blue box) shows an enlarged view of an individual cell. 100× magnification; scale bars = 10 μm. Corresponding bright-field micrographs below. (B) 3D objects analysis thresholding of A. 3D objects analysis detected LysoTracker-positive objects 10–280 voxels in size (∼500–1,500 nm diameter). (C) Mean number of LysoTracker-positive objects per cell in NTg and SORL1KO Ramos-Cas9 cell lines. (D) MFI of LysoTracker-positive objects per cell in NTg and SORL1KO Ramos-Cas9 cell lines. (E) Mean surface area of LysoTracker-positive objects per cell in NTg and SORL1KO Ramos-Cas9 cell lines. (F) Mean object volume of LysoTracker-positive objects per cell in NTg and SORL1KO Ramos-Cas9 cell lines. (G) Mean distance from center to surface of LysoTracker-positive objects per cell in NTg and SORL1KO Ramos- Cas9 cell lines. (H) Flow cytometry population histograms for unstained (orange), NTg (blue), and SORL1KO (red) I LysoTracker MFI in NTg and SORL1KO Ramos-Cas9 cell lines. (I) Quantification of LysoTracker MFI in NTg and SORL1KO Ramos-Cas9 cell lines. n = 4–6 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by different symbols). (C–G)n = 4–6, each the mean of ∼100 cells from six images from two independent experiments. Data show mean ± SEM.