memglow tm 560 probe  (Cytoskeleton Inc)

Journal: medRxiv

Article Title: Urinary vesicle biomarkers and kidney function – Results from the German AugUR study

doi: 10.64898/2026.01.12.26343937

Figure Lengend Snippet: Co-staining of the lipid bilayer of urinary extracellular vesicles with MemGlow TM 560 (purple) and the podocyte-specific marker podocalyxin with the antibody PODXL488 (cyan).

Article Snippet: In a pilot study, EVs from one young subject were characterized employing the MemGlow TM 560 probe (Cytoskeleton Inc., MG02-02), a fluorogenic probe that integrates into lipid bilayers [ ], in combination with an antibody against the podocyte-specific marker protein podocalyxin.

Techniques: Staining, Marker

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Blockage of Pathological Extracellular Vesicles and Particles From Fibroblast‐Like Synoviocytes for Osteoarthritis Relief: Proteomic Analysis and Cellular Effect

doi: 10.1002/jev2.70162

Figure Lengend Snippet: Pathogenic FLS EVPs disrupt chondrocyte and macrophages homeostasis in vitro. (A) Representative fluorescence for the internalization of EVPs by mouse chondrocytes. Scale bar = 20 µm. The red fluorescence represents EVPs labelled with the MemGlow fluorescent dye, and the blue fluorescence represents nuclei stained with DAPI. (B, C) Representative images and quantification of SA‐β‐gal staining for mouse chondrocytes after co‐culturing with different EVPs for 48 h, blue arrow indicting the SA‐β‐gal positive cells. Scale bar = 200 µm. (D) Representative Western blot images showing the senescence‐associated markers P16 and γ‐H2AX in chondrocytes treated with three types of EVPs for 48 h. (E) mRNA expression for OA‐related genes of mouse chondrocytes after co‐culturing with different EVPs for 24 h. (F–K) Representative images and quantification of COL2A1 and TUNEL staining for mouse chondrocytes, as well as EdU staining for ATDC5 cell line after stimulation with different EVPs for 48 h. Scale bar = 200 µm. (L) Internalization of EVPs by RAW264.7 macrophages. Scale bar = 200 µm. (M) mRNA expression for senescence marker ( Cdkn1a ), M1 polarization‐related genes ( Il6 , Tnf , Nos2 and Ptgs2 ), and M2 polarization‐related genes (Arg1 , Cd163 and Cd206 ) of RAW264.7 macrophages after co‐culturing with different EVPs for 24 h. (N–Q) Representative images and quantification of iNOS and P16 fluorescence staining for RAW264.7 macrophages after stimulation with different EVPs for 48 h. Scale bar = 50 µm. (R, S) Concentration of TNF‐α and IL‐6 in the cell culture supernatant of EVP‐stimulated RAW264.7 macrophages. ** Indicates p < 0.01, * indicates p < 0.05, ns indicates p > 0.05, versus the indicated groups, one‐way ANOVA.

Article Snippet: For EVP labelling and uptake studies, isolated EVPs (2 × 10 10 particles/mL in PBS) were incubated with 40 nM MemGlow dye (MG02, Cytoskeleton) at room temperature, protected from light.

Techniques: In Vitro, Fluorescence, Staining, Western Blot, Expressing, TUNEL Assay, Marker, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: Visualizing extracellular vesicle-mediated RNA transfer using a novel metabolic labeling approach

doi: 10.1101/2025.08.28.672797

Figure Lengend Snippet: (A) Western blot analysis of MDA-MB-231 cell and EV lysates comparing common EV marker s , TSG101 and CD9, organelle marker Calnexin, and Actin loading control. Equal amounts of protein were loaded for cells and EVs. (B) Size distribution of isolated MDA-MB-231 EVs as determined by NTA. Data is represented as mean ± SD (grey). (C) dSTORM image from a representative experiment showing detection of CD9 (yellow) and 5-EU (magenta) in isolated MDA-MB-231 EVs after incubation of donor cells with 5-EU. Scale bar = 1µm. Bottom right: zoomed-in view of one cluster showing coincidence of CD9 and 5-EU. Scale bar = 200nm. (D) Quantitative cluster analysis of dSTORM data. Results are expressed as the percentage of 5-EU + clusters relative to MemGlow560 + clusters and compared to EVs isolated from DMSO-treated donor cells.

Article Snippet: Isolated EVs were labeled with 200nM MemGlow560 (Cytoskeleton) for 30 minutes at RT.

Techniques: Western Blot, Marker, Control, Isolation, Incubation

Journal: bioRxiv

Article Title: Visualizing extracellular vesicle-mediated RNA transfer using a novel metabolic labeling approach

doi: 10.1101/2025.08.28.672797

Figure Lengend Snippet: (A) Overexpression of UCK2 in donor cells enhances the 5-EU to 5-EU monophosphate conversion. As a result, more 5-EU monophosphate is converted to 5-EU triphosphate by UMP-CMPK and NDPK, and a higher ethynyl labeling density and more sensitive detection is obtained using click chemistry. Created in BioRender. Vader, P. (2025) https://BioRender.com/3pwgjvv Lentiviral DNA construct engineered for stable overexpression of UCK2 in donor cells. UCK2 is tagged with a FLAG-tag enabling detection of UCK2 expression. Additionally, an internal ribosomal entry site (IRES) is encoded upstream of a blasticidin (BSD) resistance gene for generating and maintaining stable expression of UCK2. (C) Western blot analysis for FLAG and GAPDH expression in MDA-MB-231 UCK2 + cell lysate and MDA-MB-231 wildtype cell lysate. Equal amounts of protein were loaded. (D) Confocal microscopy image of MDA-MB-231 UCK2 + donor cells. FLAG expression (green) was detected through immunofluorescence, 5-EU (magenta) through click chemistry, and nuclei (cyan) using Hoechst33342. (E) Fluorescence microscopy images showing a comparison of 5-EU labeling (magenta) in MDA-MB-231 UCK2 + and MDA-MB-231 wildtype donor cells upon incubation with 5-EU for 1 hour, 2 hours, 4 hours, and 20 hours. (F) Quantification of fluorescent signal from microscopy images in E. Mean fluorescence intensity per cell is represented over time for wildtype donor cells and UCK2+ donor cells. Data is presented as mean ± SD (G) Representative dSTORM image of EVs isolated from MDA-MB-231 UCK2 + after 5-EU treatment. EVs were labeled using CD9 antibodies (yellow) and RNA was labeled using 5-EU (magenta). (H) Quantitative cluster analysis of dSTORM experiment. Data are represented as percentage of 5-EU + clusters/MemGlow560 + clusters and compared to EVs isolated from DMSO treated UCK2 + donor cells.

Article Snippet: Isolated EVs were labeled with 200nM MemGlow560 (Cytoskeleton) for 30 minutes at RT.

Techniques: Over Expression, Labeling, Construct, FLAG-tag, Expressing, Western Blot, Confocal Microscopy, Immunofluorescence, Fluorescence, Microscopy, Comparison, Incubation, Isolation

Journal: bioRxiv

Article Title: Leucine zipper-based SAIM imaging identifies therapeutic agents to disrupt the cancer cell glycocalyx for enhanced immunotherapy

doi: 10.1101/2024.12.05.627089

Figure Lengend Snippet: a , Schematic representation of constructs used to make Bzip-PDGFRβ TM constructs and Azip-Sortag-sfGFP construct. b , Schematic representation of cell membrane labelling using Azip-Bzip interaction. c , Fluorescence and bright-field images of Bzip-overexpressing Capan-2 pancreatic cancer cells with 1 µM Azip-sfGFP for 1 hour at 4°C. Scale bars, 10 µm. d , Fluorescence images of Bzip-mScarlet and Azip-sfGFP on indicated cancer cell lines for 1 hour at 4°C. Scale bars, 10 µm. e , Schematic of sortase-mediated reaction on Azip-sortag-sfGFP to replace the fluorescent protein with a small organic fluorophore. f , Coomassie blue staining analysis of sortase-mediated modification of Azip-sortag-sfGFP to yield Azip-Alexa Fluor 647 (AF647). g, h , Fluorescence and bright-field images of KPL-1 wild-type ( g ) and Bzip-overexpressing KPL-1 breast cancer cells ( h ) double labeled with Azip-AF488 and Azip-AF647 (1:100 dilution) for 1 hour at 4°C. Scale bars, 10 µm. i, Representative fluorescence microscopy of Bzip-expressing KPL-1 cells labeled with various membrane stains: Azip-AF488, Azip-AF647, DiO, CellBrite 650, or MemGlow 560 according to manufacturers’ recommended buffer compositions and concentrations. Cells were stained for 10 minutes at 37°C followed by two washes with culture medium. j , Quantification of normalized membrane fluorescence intensity over time (20 × 20 pixel regions, 2.98 × 2.98 μm) averaged from minimum 11 cells per condition. k , Membrane fluorescence intensity of each dye immediately after labelling (time 0). In k , statistical analysis was performed by one-way ANOVA with Tukey’s post hoc tests.

Article Snippet: For SAIM imaging of 1E7 cells, cells were induced with various doxycycline concentrations for 24 hours and then rinsed with serum-free, phenol-red free DMEM and incubated with MemGlow dyes (MemGlow 560, MG02-2; Cytoskeleton) in serum-free, phenol red-free DMEM for 10 minutes at 37°C.

Techniques: Construct, Membrane, Fluorescence, Staining, Modification, Labeling, Microscopy, Expressing