phage2 mag gal3 plasmid  (Addgene inc)

Journal: Journal of Extracellular Vesicles

Article Title: Development of a Live‐Cell Imaging Assay to Elucidate Spatiotemporal Dynamics of Extracellular Vesicle Fusion with Target Cells

doi: 10.1002/jev2.70228

Figure Lengend Snippet: EV‐FUSIM facilitates real‐time visualization of VSV‐G‐mediated EV‐fusion. (A) Live HeLa STAb‐GFP cells were subjected to time‐lapse imaging immediately after addition of medium only or medium containing 10 8 transfection ctl or VSV‐G EVs, taking Z‐stacks at a 15 min time interval. Images are maximum intensity projections acquired at 1 h post‐EV‐addition, showing fluorescence channels for mScar3 (top) and GFP (middle) in the same fields‐of‐view. Gamma was adjusted to 2 (mScar3) or 1.2 (GFP) for visualization purposes only. Scale bars represent 20 µm. White insets correspond to magnifications (bottom), which show mScar3, GFP and merged channels. Scale bar represents 5 µm. Images are representative of n = 3 independent experiments. (B–E) After segmentation of cells in 3D based on the GFP channel, cell‐associated fluorescent spots in the red and green channels were counted for each field‐of‐view. In tandem, the total cell volume per field‐of‐view was counted, which was used to correct spot counts for differences to the average cell volume per field‐of‐view. Graphs show the corrected mean spots per timepoint (B, D) or mean maximum spot detection over the course of the experiment (C, E) per field‐of‐view for both mScar3 and GFP channels ± SEM, calculated from n = 3 independent experiments with 2–6 fields‐of‐view per condition each. * p ≤0.05, ** p ≤0.01 and *** p ≤0.001 as determined by one‐way ANOVA with Tukey's multiple comparisons test. (F) Live HeLa STAb‐GFP were subjected to time‐lapse imaging 30 min after addition of medium containing 10 8 VSV‐G EVs, taking Z‐stacks at a 1 min time interval. Images are representative of n = 2 independent experiments. Overview image is a maximum intensity projection of the start of the experiment, showing a merge of fluorescence channels for mScar3 and GFP. Scale bar represents 20 µm. Insets shown in white correspond to magnified images of single EV‐containing endosomes on the right, showing fluorescence channels for mScar3, GFP and a merge of both at the indicated timepoints post‐EV‐addition. Scale bars represent 1 µm. (G) Live HeLa mAG‐Gal3 cells were subjected to time‐lapse imaging immediately after addition of medium only, medium containing 10 8 VSV‐G EVs or medium containing 1 mM LLOME, taking Z‐stacks at a 15 min time interval. Images are maximum intensity projections acquired at 1 h post‐EV‐addition, showing the fluorescence channel for mAG. Gamma was adjusted to 1.2 (mAG) for visualization purposes only. Scale bar represents 20 µm. (H, I) After segmentation of cells in 3D based on the mAG channel, cell‐associated fluorescent spots in the green channel were counted for each field‐of‐view. In tandem, the total cell volume per field‐of‐view was counted, which was used to correct spot counts for differences to the average cell volume per field‐of‐view. Graph shows the corrected mean spots per timepoint (H) or mean maximum spot detection over the course of the experiment (I) per field‐of‐view for the mAG channel ± SEM, calculated from n = 3 independent experiments with 5–6 fields‐of‐view per condition each. **** p≤0.0001 as determined by one‐way ANOVA with Tukey's multiple comparisons test.

Article Snippet: For the generation of endolysosomal rupture reporter cells (HeLa Gal3), a pHAGE2 mAG‐Gal3 plasmid (gift from Prof. Niels Geijsen—Addgene #62734) (D'Astolfo et al., ) for expression of mAzamiGreen‐tagged Galectin‐3, and a previously described pHR NLS‐BFP plasmid (Boersma et al., ) for tagging of the nucleus with Blue Fluorescent Protein, were used.

Techniques: Imaging, Transfection, Fluorescence