SorLA localizes to endolysosomal compartments in Ramos B cells. (A) Confocal micrograph showing surface and intracellular SorLA-EGFP localization in Ramos cells. 100× magnification; inset (blue box) shows an enlarged view of an individual cell. Scale bars = 10 µm. (B) Representative flow cytometry histograms for control secondary antibody only stain (orange), unpermeabilized cell surface SorLA stain (red), and 0.01% Triton X-100 permeabilized total SorLA stain (blue). (C) Surface SorLA expression as % of total by flow cytometry. Surface SorLA expression = (surface SorLA MFI/total SorLA MFI) × 100. n = 9 independent Ramos-Cas9 transductions with three NTg sgRNAs (indicated by symbols) from three independent experiments. Data show mean ± SEM. (D) Quantification of SorLA-EGFP colocalization with organelle markers. Voxel-based Manders’ coefficient. Rab5: n = 31 cells, Rab11: n = 90 cells, Rab7: n = 60 cells, and LAMP1: n = 41 cells from 10 images from two independent experiments. (E) Quantification of endogenous SorLA colocalization with organelle markers at 5, 15, 30, and 60 min after addition to PMS presenting anti-IgM surrogate antigen. Manders’ coefficient. Rab5: n = 13–31 cells, Rab11: n = 7–29 cells, Rab7: n = 7–31 cells, and LAMP1: n = 27–58 cells from four independent experiments. These intracellular compartments are not mutually exclusive; thus, SorLA puncta may be positive for several markers and be counted under >1 organelle. Data are shown as box and whisker plots, where the line indicates the median, the box represents the interquartile range, and whiskers denote the minimum and maximum values. (F) Representative fluorescence micrographs of Ramos-Cas9 cells on PMS presenting anti-IgM surrogate antigen at 15 min. Images show SorLA (magenta), vesicle markers (cyan), and an overlay from a single z-stack slice (ImageJ). “3D” column shows a 3D projection of the overlay z-stack (Imaris). Colocalization appears white.