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: FLS pathology was accompanied by increased EVP secretion in OA. (A) Gene ontology (GO) cellular component enrichment analysis of differentially expressed genes between normal and OA synovium. (B, C) Violin plots for the expression levels of the exosome secretion marker (RAB27A), senescence markers (CDKN1A, CDKN2A) and inflammation‐related markers (PTGS2, IL6, MMP13), along with Pearson correlation analyses between the expression levels of these markers and RAB27A. Data was normalized as fragments per kilobase of exon model per million mapped fragments (FPKM). (D–G) Multiplex immunohistochemical (mIHC) staining of normal and OA synovium, with magnified views of the boxed areas showing individual colour channels. Scale bar = 100 µm. Line graphs display the relative intensity and co‐localization of each fluorescence along the line in the magnified images. (H, I) Representative RAB27A fluorescence staining images and quantification in the synovial region of sham‐operated mice and mice at 4 and 8 weeks post‐DMM surgery. Scale bar = 25 µm. (J) Schematic diagram of in vitro EVP collection. Briefly, FLSs were pre‐treated with IL‐1β (10 ng/mL) and Bleomycin (25 µg/mL) for 24 h and changed into fresh medium without inducers. EVPs were then isolated from the conditioned medium through ultracentrifugation. (K, L*) Representative MMP13 fluorescence staining and SA‐β‐gal staining images for FLSs after inflammation and senescence induction. (M) Diameter distribution of EVPs from control and pathological FLSs by NTA, with screenshots of the particle flow. (N, O) Representative TEM images for EVPs and Western blot gels for EVP protein markers. Scale bar = 200 µm. (P, Q) Quantification and protein concentration of EVPs isolated from different culture medium, both of which were normalized to original cell counts. ** 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: Expressing, Marker, Multiplex Assay, Immunohistochemical staining, Staining, Fluorescence, In Vitro, Isolation, Control, Western Blot, Protein Concentration

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: Proteomic profiling of pathogenic EVPs reflected the pathological changes of the source cells. (A) Schematic diagram for proteomics analysis of EVPs isolated from FLSs after inducing inflammatory and senescent phenotypes, created with Figdraw ( www.figdraw.com ). (B) SDS‐PAGE gel electrophoresis images of proteins lysed from abovementioned EVPs. (C) Number of proteins identified by mass spectrometry in EVPs secreted from control FLSs and FLSs induced with inflammation and senescence. (D, E) Principal component analysis plot and Pearson's Correlation Coefficient heatmap of the protein composition in EVPs from three groups ( n = 3 samples per group). The gradient colours and annotated values represent the Pearson correlation coefficients. (F) Number of differentially expressed proteins in EVPs between the three groups, with screening criteria set at an adjusted p value < 0.01 and a fold change >2 or <0.5. (G) Circos plot visualization of the overlaps among significantly altered proteins that overlap in Inf‐EVP and Sen‐EVP, with lines connecting the commonly altered proteins. (H) Pathway heatmaps of significantly differentially expressed proteins in Inf‐EVP and Sen‐EVP compared to Ctr‐EVP, enriched using Metascape. (I, J) Representative gene set enrichment analysis of proteins in Inf‐EVP or Sen‐EVP relative to those in Ctr‐EVP.

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: Isolation, SDS Page, Nucleic Acid Electrophoresis, Mass Spectrometry, Control

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: 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 impair chondrogenic differentiation of mesenchymal stem cells. (A) Schematic diagram of EVP stimulation on mouse BMSCs isolated from the femoral bone marrow cavity of mice. (B) Internalization of EVPs by BMSCs. Scale bar = 50 µm. (C) Expression of chondrogenic differentiation‐related genes in chondrogenesis‐induced BMSCs after 7 days of treatment with different EVPs. (D) Representative Western blot images of COL10A1, the marker of chondrocyte hypertrophy. (E, F) Representative images of alcian blue staining and SA‐β‐gal staining in BMSCs after 7 days of stimulation with different EVPs. Scale bar = 1 mm and 200 µm separately. (G) Schematic diagram of section staining observation after inducing BMSCs to form chondrocyte pellets for 21 days while simultaneously stimulating with different EVPs. (H, I) Representative images of SOX9 fluorescence staining, safranin O (SO) staining, alcian blue (AB) staining and toluidine blue (TB) staining of BMSC‐differentiated chondrocyte pellet sections. Scale bar = 50 µm. (J) Schematic diagram of EVP treatment on mouse ADSCs isolated from the iWAT of mice. (K) Internalization of EVPs by ADSCs. Scale bar = 50 µm. (L, M) Representative images of SA‐β‐gal staining and alcian blue staining of ADSCs after 7 days of chondrogenic induction and treatment with different EVPs. (N) Representative images of SOX9 staining in sections of chondrocyte pellets formed by ADSC after 21‐day induction.

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: Isolation, Expressing, Western Blot, Marker, Staining, Fluorescence

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: Intra‐articular injection of FLS‐targeting AAV for delivering Rab27a ‐shRNA to specifically reduce EVP secretion. (A) Rab27a expression levels in FLSs after transfection with control shRNA and Rab27a knockdown shRNA. (B, C) EVP size distribution and quantification after transfection with control shRNA and Rab27a knockdown shRNA. ** Indicates p < 0.01, versus the indicated groups, student’s t ‐test. (D) Representative Western blot images for positive and negative surface markers of EVPs at equal concentration after shRNA transfection. (E) Schematic diagram of constructing a virus targeting FLS to inhibit Rab27a for intra‐articular injection. The synovium‐affinity peptide HAP‐1 is fused with the AAV9 viral capsid viral protein 2 (VP2), and the AAV9 vector is designed to simultaneously carry the gene encoding the mScarlet fluorescent protein and an expression cassette for shRNA. (F) Representative images of mScarlet fluorescence in the synovium and cartilage regions of mouse knee joints. Scale bar = 200 µm and 100 µm separately. (G) Representative images of mScarlet fluorescence in sections of multiple mouse organs. Scale bar = 200 µm. (H) Representative images and quantitative results of RAB27A expression in the synovial region after intra‐articular AAV injection. ** Indicates p < 0.01, versus the indicated groups, two‐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: Injection, shRNA, Expressing, Transfection, Control, Knockdown, Western Blot, Concentration Assay, Virus, Plasmid Preparation, Fluorescence

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