Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Factors to consider before choosing EV labeling method for fluorescence-based techniques

doi: 10.3389/fbioe.2024.1479516

Figure Lengend Snippet: Common label types.

Article Snippet: MemGlow (MG) , BioActs , membrane-specific , bright fluorescence, minimal background; creates non-fluorescent aggregates, allows the detection of short/transient interactions , potential photobleaching, possible dye transfer through brief interaction with the recipient cell , may affect EV uptake and cargo distribution , EV membrane labeling , .

Techniques: Staining, Membrane, Labeling, Live Cell Imaging, Binding Assay, Functional Assay, In Vitro, Fluorescence, Imaging, In Vivo, In Vivo Imaging, Solubility, Concentration Assay, Liposomes, Microscopy, Flow Cytometry, Permeability, Modification, Disruption, Avidin-Biotin Assay, Isolation, Expressing

Journal: bioRxiv

Article Title: Biofunctional matrix models reveal mineral-dependent mechanoregulation of bone metastatic breast cancer

doi: 10.1101/2022.06.29.498001

Figure Lengend Snippet: a, Schematic of integrin-mediated mechanosignaling and effect of different mechanosignaling inhibitors on the growth of MDA-MB231 cells cultured on collagen and mineralized collagen ( n = 4). b, Traction force microscopy of MDA-MB231 cells following preculture on collagen and mineralized collagen. Representative images of MemGlow 590-labeled cells (top), traction maps (middle), and corresponding quantification of traction forces (bottom). Scale bar = 50 µm. P < 0.05 by Mann-Whitney U-test. c, Traction force microscopy of GFP high and GFP low MDA-MB231 cells. Representative fluorescence images (top), traction maps (middle), and corresponding quantification of traction forces (bottom) of Nanog-GFP cells. White lines on traction maps indicate contour of cell. Scale bar = 50 µm. P < 0.05 by Mann-Whitney U-test. *: P < 0.05, **: P < 0.01.

Article Snippet: Confocal microscopy (810 and 880 LSM, Zeiss, Germany) was used to image Nanog-GFP or MemGlow 590 (20 nM, Cytoskeleton, US) labeled MDA-MB231 cells using either 63× or 40×,1.2NA C-Apochromat objectives.

Techniques: Cell Culture, Microscopy, Labeling, MANN-WHITNEY, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Neuron‐derived extracellular vesicles contain synaptic proteins, promote spine formation, activate TrkB‐mediated signalling and preserve neuronal complexity

doi: 10.1002/jev2.12355

Figure Lengend Snippet: Neuron‐derived EVs are taken up both in the soma and dendrites and their internalization is mediated by clathrin‐dependent endocytosis. Cultured neurons were transfected at DIV12 with an eGFP plasmid (in green). EVs were isolated from sister neuronal cultures and labeled with MemGlow‐560 (in red). (A) Orthogonal views show EVs‐uptake in the soma, in dendrites, and also in dendritic spines. The graph shows the percentage of internalized EVs in the soma and in the dendrites. (B) Memglow‐560 specifically labels EVs, as when used to label non‐EVs samples (only 1X PBS) there was no fluorescent signal. (C) Experimental design to assess the internalization mechanism of EVs in recipient neurons. EVs used for treatment were isolated from sister cultured neurons at DIV13. eGFP‐transfected neurons were treated with uptake‐inhibitors CPZ (5 µM) and Dyn (80 µM) 30 min prior to EVs treatment. (D) Representative images showing EVs‐uptake in neurons. Images in the z‐stack are processed with Image‐J as Z‐Projects. Images below show the colocalization dots between both channels, which represent EVs that have been internalized by neurons. The graphs show the percentage of internalized EVs per neuron. Ø = No treatment; CPZ = Chlorpromazine; Vehicle = DMSO; Dyn = Dynasore. Scale bar of 5 µm in all the images. Values represent different neurons (≥ 5 per experiment) from three independent neuronal cultures (mean ± SEM). Data was analysed by Student's t ‐test (**** P < 0.0001 vs. soma, * P < 0.05 vs. Veh, ** P < 0.01 vs. Ø).

Article Snippet: For the EVs uptake analysis, EVs were labelled with MemGlow‐560 (Cytoskeleton Inc.), as previously described (Boyer et al., ).

Techniques: Derivative Assay, Cell Culture, Transfection, Plasmid Preparation, Isolation, Labeling