pkh67-labeled sevs  (Tocris)

Journal: Bioactive Materials

Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

doi: 10.1016/j.bioactmat.2026.01.002

Figure Lengend Snippet: Generation of bioengineered LEVs (LEVs@TA) through in situ TA modifications. ( A ) Schematic illustration showing the interaction of TA with the phospholipid bilayer of LEVs via hydrogen bonding and the uptake of LEVs@TA by macrophages. ( B ) The percentages of CY5-TA-modified cells following incubation with gradient concentrations of CY5-TA (0, 0.1, 1, 5, and 10 μM) for 24 h (flow cytometry assay). ( C ) Colocalization of CY5-TA on HEK293T cells following incubation in 10 μM CY5-TA for 24 h (fluorescence microscopy). The nuclei were stained with DAPI (blue). ( D ) The percentages of CY5-TA-modified LEVs following incubation with gradient concentrations of CY5-TA (0, 10, 20, 50, and 100 μM) for 24 h (flow cytometry assay). ( E ) Colocalization of CY5-TA and PKH67-labeled LEVs (green) following incubation with 100 μM CY5-TA for 24 h (fluorescence microscopy). ( F ) Snapshots of CGMD simulations depicting the uptake of LEVs and LEVs@TA by macrophages at 0, 5, 10, 15, and 20 ns. ( G ) Representative in vivo fluorescence images showing good stability of DIO-labeled-LEVs@CY5-TA in vivo .

Article Snippet: PKH67-labeled SEVs and LEVs resuspended in complete medium were used to treat RAW 264.7 cells for 24 h. The cells were then fixed with 4 % PFA (Coolaber), permeabilized, and stained for cytoskeletal visualization using fluorescein phalloidin (1:1000 dilution; MCE) for 30 min.

Techniques: In Situ, Modification, Incubation, Flow Cytometry, Fluorescence, Microscopy, Staining, Labeling, In Vivo

Journal: Bioactive Materials

Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

doi: 10.1016/j.bioactmat.2026.01.002

Figure Lengend Snippet: Endo/lysosomal escape capacity of bioengineered LEVs@TA following uptake by macrophages. ( A ) Schematic illustration showing the endo/lysosomal escape process of LEVs@TA within the cytoplasm of macrophages. After uptake by macrophages, LEVs@TA were entrapped within endo/lysosomes, and then TA underwent protonation and disassembled from LEVs in an acidic environment, leading to rupture of the endo/lysosomal structure. ( B ) Snapshots of CGMD simulations showing the disassembly of TA and LEVs in the lysosomal environment. ( C ) Colocalization of LysoTracker-labeled endo/lysosomes (violet) and PKH67-labeled LEVs or LEVs@TA (green) (fluorescence microscopy). The nuclei were stained with Hoechst (blue). ( D ) Quantification of the colocalization of endo/lysosomes and LEVs or LEVs@TA using the Pearson correlation coefficient ( n = 12). ( E ) Schematic illustration showing the leakage of calcein into the cytosol when TA diffused from LEVs@TA and destabilized the endo/lysosomal membranes. ( F ) The distribution of calcein (green) in macrophages treated with PBS, LEVs, and LEVs@TA (fluorescence microscopy). (G) Representative TEM images of macrophages showing the structure of lysosomes in macrophages treated with LEVs and LEVs@TA. The data are expressed as the mean ± SEM. Statistical analysis was performed with Student's t -test ( D ). ∗∗∗ p < 0.001 indicates significant differences between the indicated columns.

Article Snippet: PKH67-labeled SEVs and LEVs resuspended in complete medium were used to treat RAW 264.7 cells for 24 h. The cells were then fixed with 4 % PFA (Coolaber), permeabilized, and stained for cytoskeletal visualization using fluorescein phalloidin (1:1000 dilution; MCE) for 30 min.

Techniques: Labeling, Fluorescence, Microscopy, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Tumor-associated macrophage-derived exosomal miR21-5p promotes tumor angiogenesis by regulating YAP1/HIF-1α axis in head and neck squamous cell carcinoma

doi: 10.1007/s00018-024-05210-6

Figure Lengend Snippet: TAMs-EVs promote angiogenesis of HUVECs in vitro. A IF image demonstrates that HUVECs have taken up and internalized PKH67-labeled sEVs derived from macrophages, Scale bars = 50 μm. B Representative micrographs of tube formation assay induced by M0/TAM-sEVs, Scale bars = 100 μm. C The numbers of branch points were calculated by ImageJ. D Schematic overview (left) and channel configuration (right) of the microfluidic chip designed for the induction of angiogenesis. E Physical diagram of a microfluidic chip compared with a one-yuan Chinese currency coin. F Images of vascular sprouts induced by M0/TAM-sEVs, Scale bars = 100 μm. G , H Quantitative analyses of angiogenesis in terms of average sprout length and number. ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: M0/TAM-sEV labeled with PKH67 (Thermo Fisher Scientific) were incubated with HUVECs for 6 h. To visualize the HUVECs, DAPI (Solarbio, C0065, Beijing, China) was used to stain the nucleus, and rhodamine-labeled phalloidin (Yeasen, 40734ES75, Shanghai, China) was used to label the cell cytoskeleton.

Techniques: In Vitro, Labeling, Derivative Assay, Tube Formation Assay

Journal: International Journal of Oral Science

Article Title: Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling

doi: 10.1038/s41368-023-00236-1

Figure Lengend Snippet: Collagen crosslinking triggered by CAF sEV-LOX. a Internalization of CAF-S1/S2/S3/S4 sEV (green) by cells. Normal fibroblasts (NFs) were cultured for 12 h ( n = 3 per group). sEVs were mostly internalized into NFs (red) in large numbers (arrows). Small numbers of sEVs bound to the ECM (arrowheads). Left, representative images. (Scale bar = 10 µm). Right, quantification results. b NFs (red) were cultured for 72 h ( n = 3 per group). Adhesion of CAF-S1/S2/S3/S4 sEV (green) to ECM (arrowheads). Left, representative images. (Scale bar = 10 µm). Right, quantification results. c ELISA assay of PYD, DHLNL, HLNL. NFs were cultured for 72 h, then treated with CAF-S1/S2/S3/S4 sEV with or without anti-LOX antibody or BAPN ( n = 3 per group). PBS was used as a negative control ( n = 3 per group) and glucose ( n = 3 per group) was used as a positive control. ns , not significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Then PKH67-labeled sEVs (20 µg per well) were added into the collagen I-containing wells and incubated at 37 °C for 12 h. To inhibit integrin α2β1-mediated collagen binding, sEVs were pre-incubated with 2 µM TCI-15 (TOCRIS, Oxfordshire, UK) at 37 °C for 1 h. After washing with PBS, images were recorded by an inverted fluorescent microscope (Olympus IX71).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control

Journal: International Journal of Oral Science

Article Title: Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling

doi: 10.1038/s41368-023-00236-1

Figure Lengend Snippet: Collagen binding of CAF sEVs via integrin α2β1. a Schematic of collagen detection in CAF sEVs via the surface receptor. b Western blot analysis of integrin β1, integrin α2, and integrin α4 expression in CAF-S1/S2/S3/S4 and their sEVs. CD9 and HSP70 were used as CAF sEV markers. c Inhibition of the binding of CAF-S1/S2/S3/S4 sEV (green) to collagen I by treatment with TC I-15 in vitro ( n = 3 per group). Left, representative images. (Scale bar = 50 µm). Right, quantification results. d Representative TEM images of collagen crosslinking induced by CAF-S1/S2 sEV with or without TC I-15 treatment. PBS was used as a control. Thick collagen fibers (arrows) and thin collagen fibers (arrowheads) associated with sEVs were observed. (Scale bar = 100 nm). e ELISA assay of PYD, DHLNL, HLNL levels in the collagen matrix treated with CAF-S1/S2/S3/S4 sEV with or without TC I-15 ( n = 3 per group). For blots source data, see Fig. . ns , not significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Then PKH67-labeled sEVs (20 µg per well) were added into the collagen I-containing wells and incubated at 37 °C for 12 h. To inhibit integrin α2β1-mediated collagen binding, sEVs were pre-incubated with 2 µM TCI-15 (TOCRIS, Oxfordshire, UK) at 37 °C for 1 h. After washing with PBS, images were recorded by an inverted fluorescent microscope (Olympus IX71).

Techniques: Binding Assay, Western Blot, Expressing, Inhibition, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Oral Science

Article Title: Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling

doi: 10.1038/s41368-023-00236-1

Figure Lengend Snippet: EMT of OSCC cells induced by CAF sEVs in vitro. a Illustration of collagen crosslinking in collagen I-Matrigel mixture triggered by CAF sEVs and drive EMT of OSCC. b The morphology (phalloidin) and expression of E-cadherin, N-cadherin and vimentin in UM-SCC6 spheroids stimulated by CAF-S2/S4 sEV with or without BAPN ( n = 3 per group). PBS was used as a control ( n = 3 per group). Left: representative images. (Scale bar = 10 µm). Right: quantitative analyses of cell invasion (phalloidin) and the expression of E-cadherin, N-cadherin and vimentin in UM-SCC6 spheroids under different experimental conditions ( n = 3 per group). c Western blot analysis of E-cadherin, N-cadherin and vimentin in UM-SCC6 spheroids under different experimental conditions ( n = 3 per group). d ELISA of PYD, DHLNL, HLNL in the collagen I-Matrigel matrix treated with CAF-S2/S4 sEV with or without BAPN ( n = 3 per group). For blots source data, see Fig. . ns , not significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Then PKH67-labeled sEVs (20 µg per well) were added into the collagen I-containing wells and incubated at 37 °C for 12 h. To inhibit integrin α2β1-mediated collagen binding, sEVs were pre-incubated with 2 µM TCI-15 (TOCRIS, Oxfordshire, UK) at 37 °C for 1 h. After washing with PBS, images were recorded by an inverted fluorescent microscope (Olympus IX71).

Techniques: In Vitro, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Oral Science

Article Title: Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling

doi: 10.1038/s41368-023-00236-1

Figure Lengend Snippet: Schematic of collagen crosslinking triggered by CAF sEVs leading to EMT of OSCC. CAFs in the tumor microenvironment secrete sEVs enriched in α-LOX, which interact with FN, POSTN, and BMP-1. CAF sEVs detect collagen via integrin α2β1 and induce direct collagen crosslinking. The crosslinked matrix activates p-FAK/p-paxillin pathway. Rho/ROCK regulates actomyosin contraction, and then leads to the nuclear translocation of YAP, which in turn promotes EMT of OSCC cells

Article Snippet: Then PKH67-labeled sEVs (20 µg per well) were added into the collagen I-containing wells and incubated at 37 °C for 12 h. To inhibit integrin α2β1-mediated collagen binding, sEVs were pre-incubated with 2 µM TCI-15 (TOCRIS, Oxfordshire, UK) at 37 °C for 1 h. After washing with PBS, images were recorded by an inverted fluorescent microscope (Olympus IX71).

Techniques: Translocation Assay

Journal: International Journal of Nanomedicine

Article Title: Comparison of Curative Effect of Human Umbilical Cord-Derived Mesenchymal Stem Cells and Their Small Extracellular Vesicles in Treating Osteoarthritis

doi: 10.2147/IJN.S336062

Figure Lengend Snippet: Characterization of hUC-MSC-sEVs. ( A ) Schematic diagram of the extraction process of hUC-MSC-sEVs. ( B ) Under the electron microscope, the hUC-MSC-sEVs show a circular bilayer structure with a diameter of about 100 nm. ( C ) The results of nFCM showed that the diameter of hUC-MSC-sEVs was about 80.48 nm. ( D ) Proportional relationship among original supernatant volume, quantification of cells and vesicles particles, and amount of protein extracted from hUC-MSC-sEVs. ( E ) The surface markers of hUC-MSC-sEVs were identified by nFCM. CD9, CD63, and CD81 were found to be positive in hUC-MSC-sEVs. ( F ) hUC-MSC-sEVs’ internalization to chondrocytes. hUC-MSC-sEVs (labeled with PKH67 dye, green) and chondrocytes (nuclei were stained with DAPI) were co-incubated for 12 h, respectively. In the control group, PKH67 dye was co-incubated with chondrocytes for 12 h, respectively. Representative fluorescence images are shown above (scale bar = 100 μm; scale bar in magnification = 50 μm).

Article Snippet: Subsequently, PKH67-labeled sEVs were co-cultured with chondrocytes (CP-R092, Procell Life Sci & Tech Co. Ltd., Wuhan, China) for 12 h, and the internalization of sEVs was evaluated using fluorescence microscopy (Leica DMi8 S, Germany).

Techniques: Microscopy, Labeling, Staining, Incubation, Fluorescence