Figure 3

Quantification of eGFP-LC3B + vesicles using high throughput flow cytometry. (A), Schematic overview of the method. eGFP-LC3B expressing cell lines are permeabilized with 0.05% saponin containing buffer, the non-membrane bound eGFP-LC3B is washed out, followed by fixation with 4% PFA. Using FACS, the amount of membrane-bound eGFP-LC3B per cell is quantified as eGFP mean fluorescent intensity (MFI). (B), Forward and side scatter of HeLa eGFP-LC3B cells in flow cytometry before (blue) and after saponin treatment (red) (top panel). eGFP-LC3B fluorescence is decreased after saponin treatment of HeLa eGFP-LC3B cells (bottom panel). (C), Total eGFP-LC3B MFI of HeLa eGFP-LC3B cells, either mock-treated or treated with Rapamycin (2 µM, 4 h) (top panel). eGFP-LC3B MFI of HeLa eGFP-LC3B cells, either mock-treated or treated with Rapamycin (2 µM, 4 h) after saponin permeabilization and cytoplasmic washout of the cells (bottom panel). Data are shown as mean (n = 6) ± SEM. (D), HeLa eGFP-LC3B cells were either mock-treated or treated with, Chloroquine (10 µM) or Rapamycin (1 µM) for 4 h and subsequently saponin treated. eGFP MFIs were quantified, either in non-fixed (green), methanol fixed (blue), or paraformaldehyde (PFA) fixed cells (red). The left panel shows the raw MFI, the right panel shows the background adjusted values. Data are shown as mean (n = 3) ± SEM. Statistical significance was assessed using unpaired t-tests (C) or one-way ANOVA (D). ns = not significant, **p ≤ 0.01, ***p ≤ 0.001.