Journal: Nature Methods

Article Title: Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy

doi: 10.1038/s41592-024-02297-4

Figure Lengend Snippet: a , Emission spectrum of the membrane lipid packing probe NR12A before and after 30 confocal imaging frames (561 nm excitation) in POPC SLBs. Mean and s.d. of six measurements in three independent experiments is shown. b , Ratiometric GP images of live cells shows a change in the measured lipid packing when using standard dyes (NR12A), which is not detected when using an exchangeable fluorophore (NR4A, PAINT mode). On the right are lipid packing images after 30 frames for the standard and exchangeable fluorophore, respectively. c , Emission spectra of the exchangeable probe NR4A, conditions as in a . d , Biased increase in apparent lipid packing of the plasma membrane of Ptk2 cells is measured when using the standard probe NR12A due to photoblueing, which is avoided with the exchangeable fluorophore NR4A. Shown is the relative change of GP value on confocal excitation. Mean and s.d. of three independent measurements are shown. Excitation power was 5 µW (561 nm) at the sample plane in all cases. Scale bars, 1 µm.

Article Snippet: Fluorescent dyes and their conjugated versions were acquired from the following manufacturers: Abberior STAR RED goat antimouse IgG (Abberior, STRED-1001), Abberior STAR RED DPPE (Abberior, STRED-0200), Abberior STAR RED Streptavidin (STRED-0120), ATTO 647N free acid (ATTO-TEC, AD 647N-21), Anti-Mouse-IgG—Atto 647N antibody produced in goat (Sigma-Aldrich, 50185), ATTO 647N-DPPE (ATTO-TEC, AD 647N-151), ATTO 655 free acid (ATTO-TEC, AD 655-21), Anti-Mouse IgG—Atto 655 antibody produced in goat (Sigma-Aldrich, 50283), ATTO 655-DPPE (ATTO-TEC, AD 655-151), MemGlow NR12A Membrane Polarity Probe (Cytoskeleton, Inc., MG07), MemGlow NR4A Membrane Polarity Probe (Cytoskeleton, Inc., MG06), Abberior STAR 600-DPPE (Abberior), TopFluor Cholesterol (Avanti Polar Lipids, 810255) and ATTO 488-DPPE (ATTO-TEC, AD 488-151).

Techniques: Membrane, Imaging, Clinical Proteomics

Journal: Nature Methods

Article Title: Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy

doi: 10.1038/s41592-024-02297-4

Figure Lengend Snippet: a , Changes in the apparent membrane lipid packing as determined from the signal GP ratio of the environment-sensitive fluorophores NR12A (Standard) and NR4A (Exchangeable) in POPC SLBs membrane patches upon confocal and STED microscopy imaging (640 nm excitation, 775 nm STED) for repeated imaging frames. b , Intensity changes for channel 1 (580–630 nm), channel 2 (650–700 nm), and background intensity and subsequent changes in apparent packing as measured for NR12A in POPC SLBs membrane patches. c , Measurements as in a but with unbiased membrane lipid packing values recovered upon addition of fresh dye to the sample after several frames of STED microscopy imaging. Mean and standard deviation of three independent experiments are shown.

Article Snippet: Fluorescent dyes and their conjugated versions were acquired from the following manufacturers: Abberior STAR RED goat antimouse IgG (Abberior, STRED-1001), Abberior STAR RED DPPE (Abberior, STRED-0200), Abberior STAR RED Streptavidin (STRED-0120), ATTO 647N free acid (ATTO-TEC, AD 647N-21), Anti-Mouse-IgG—Atto 647N antibody produced in goat (Sigma-Aldrich, 50185), ATTO 647N-DPPE (ATTO-TEC, AD 647N-151), ATTO 655 free acid (ATTO-TEC, AD 655-21), Anti-Mouse IgG—Atto 655 antibody produced in goat (Sigma-Aldrich, 50283), ATTO 655-DPPE (ATTO-TEC, AD 655-151), MemGlow NR12A Membrane Polarity Probe (Cytoskeleton, Inc., MG07), MemGlow NR4A Membrane Polarity Probe (Cytoskeleton, Inc., MG06), Abberior STAR 600-DPPE (Abberior), TopFluor Cholesterol (Avanti Polar Lipids, 810255) and ATTO 488-DPPE (ATTO-TEC, AD 488-151).

Techniques: Membrane, Microscopy, Imaging, Standard Deviation

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: Nature Methods

Article Title: Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy

doi: 10.1038/s41592-024-02297-4

Figure Lengend Snippet: a , Emission spectrum of the membrane lipid packing probe NR12A before and after 30 confocal imaging frames (561 nm excitation) in POPC SLBs. Mean and s.d. of six measurements in three independent experiments is shown. b , Ratiometric GP images of live cells shows a change in the measured lipid packing when using standard dyes (NR12A), which is not detected when using an exchangeable fluorophore (NR4A, PAINT mode). On the right are lipid packing images after 30 frames for the standard and exchangeable fluorophore, respectively. c , Emission spectra of the exchangeable probe NR4A, conditions as in a . d , Biased increase in apparent lipid packing of the plasma membrane of Ptk2 cells is measured when using the standard probe NR12A due to photoblueing, which is avoided with the exchangeable fluorophore NR4A. Shown is the relative change of GP value on confocal excitation. Mean and s.d. of three independent measurements are shown. Excitation power was 5 µW (561 nm) at the sample plane in all cases. Scale bars, 1 µm.

Article Snippet: Fluorescent dyes and their conjugated versions were acquired from the following manufacturers: Abberior STAR RED goat antimouse IgG (Abberior, STRED-1001), Abberior STAR RED DPPE (Abberior, STRED-0200), Abberior STAR RED Streptavidin (STRED-0120), ATTO 647N free acid (ATTO-TEC, AD 647N-21), Anti-Mouse-IgG—Atto 647N antibody produced in goat (Sigma-Aldrich, 50185), ATTO 647N-DPPE (ATTO-TEC, AD 647N-151), ATTO 655 free acid (ATTO-TEC, AD 655-21), Anti-Mouse IgG—Atto 655 antibody produced in goat (Sigma-Aldrich, 50283), ATTO 655-DPPE (ATTO-TEC, AD 655-151), MemGlow NR12A Membrane Polarity Probe (Cytoskeleton, Inc., MG07), MemGlow NR4A Membrane Polarity Probe (Cytoskeleton, Inc., MG06), Abberior STAR 600-DPPE (Abberior), TopFluor Cholesterol (Avanti Polar Lipids, 810255) and ATTO 488-DPPE (ATTO-TEC, AD 488-151).

Techniques: Membrane, Imaging, Clinical Proteomics

Journal: Nature Methods

Article Title: Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy

doi: 10.1038/s41592-024-02297-4

Figure Lengend Snippet: a , Changes in the apparent membrane lipid packing as determined from the signal GP ratio of the environment-sensitive fluorophores NR12A (Standard) and NR4A (Exchangeable) in POPC SLBs membrane patches upon confocal and STED microscopy imaging (640 nm excitation, 775 nm STED) for repeated imaging frames. b , Intensity changes for channel 1 (580–630 nm), channel 2 (650–700 nm), and background intensity and subsequent changes in apparent packing as measured for NR12A in POPC SLBs membrane patches. c , Measurements as in a but with unbiased membrane lipid packing values recovered upon addition of fresh dye to the sample after several frames of STED microscopy imaging. Mean and standard deviation of three independent experiments are shown.

Article Snippet: Fluorescent dyes and their conjugated versions were acquired from the following manufacturers: Abberior STAR RED goat antimouse IgG (Abberior, STRED-1001), Abberior STAR RED DPPE (Abberior, STRED-0200), Abberior STAR RED Streptavidin (STRED-0120), ATTO 647N free acid (ATTO-TEC, AD 647N-21), Anti-Mouse-IgG—Atto 647N antibody produced in goat (Sigma-Aldrich, 50185), ATTO 647N-DPPE (ATTO-TEC, AD 647N-151), ATTO 655 free acid (ATTO-TEC, AD 655-21), Anti-Mouse IgG—Atto 655 antibody produced in goat (Sigma-Aldrich, 50283), ATTO 655-DPPE (ATTO-TEC, AD 655-151), MemGlow NR12A Membrane Polarity Probe (Cytoskeleton, Inc., MG07), MemGlow NR4A Membrane Polarity Probe (Cytoskeleton, Inc., MG06), Abberior STAR 600-DPPE (Abberior), TopFluor Cholesterol (Avanti Polar Lipids, 810255) and ATTO 488-DPPE (ATTO-TEC, AD 488-151).

Techniques: Membrane, Microscopy, Imaging, Standard Deviation

Journal: Scientific Reports

Article Title: A 3D adrenocortical carcinoma tumor platform for preclinical modeling of drug response and matrix metalloproteinase activity

doi: 10.1038/s41598-023-42659-0

Figure Lengend Snippet: A depiction of the overall workflow of the study. NCI-H295R ACC cells are used either as traditional 2D cell cultures or ECM hydrogel-based 3D tumor constructs. The culture models were then characterized through histology and IF and deployed within chemotherapy screens. The 3D constructs were also functionalized with MMP-sensitive fluorogenic peptide biosensors to evaluate cellular MMP activity within the 3D ECM hydrogel, or implemented within a metastasis-on-a-chip platform.

Article Snippet: NCI-H295R cells were fluorescently tagged using MemGlow 560 (Cytoskeleton, Inc., Denver, CO) following manufacturer instructions.

Techniques: Construct, Activity Assay

Journal: Scientific Reports

Article Title: A 3D adrenocortical carcinoma tumor platform for preclinical modeling of drug response and matrix metalloproteinase activity

doi: 10.1038/s41598-023-42659-0

Figure Lengend Snippet: ACC 3D constructs demonstrate MMP activity. ( a ) Light induced thiol-ene bonds between the thiolated HA, thiol group in the MMP biosensor, and methacrylate groups on collagen. ( b ) Biosensor sensing of cell MMP activity in ECM hydrogels using live-cell confocal imaging. (i) Light microscopy of cells within hydrogels. (ii) Fluorescent imaging of the NCI-H295R cell labeled with a DiD cell membrane intercalating dye. (iii) MMP biosensor is visualized by the white signal. ( c ) MMP activity measured in NCI-H295R ACC cell line 3D ECM cultures and with the GM6001 MMP inhibitor. Statistical significance: *p < 0.05.

Article Snippet: NCI-H295R cells were fluorescently tagged using MemGlow 560 (Cytoskeleton, Inc., Denver, CO) following manufacturer instructions.

Techniques: Construct, Activity Assay, Imaging, Light Microscopy, Labeling, Membrane

Journal: Scientific Reports

Article Title: A 3D adrenocortical carcinoma tumor platform for preclinical modeling of drug response and matrix metalloproteinase activity

doi: 10.1038/s41598-023-42659-0

Figure Lengend Snippet: MMP activity is required for invasion. ( a ) Microfluidic devices were designed with four A549 lung epithelial cell-containing hydrogels and a circulating fluorescently-tagged NCI-H295R population. ( b ) ACC cells were circulated through the devices for 4 days either with or without GM6001 MMP inhibitor. ( c ) MMP inhibition did not significantly affect the number of NCI-H295R ACC cells that were able to attach to and invade the A549 lung hydrogels, however the average distance the ACC cells invaded into the A549 lung hydrogels was significantly reduced with MMP inhibition. Representative images demonstrate the disparity in invasion distance between conditions. Statistical significance: *p < 0.01.

Article Snippet: NCI-H295R cells were fluorescently tagged using MemGlow 560 (Cytoskeleton, Inc., Denver, CO) following manufacturer instructions.

Techniques: Activity Assay, Inhibition

Journal: bioRxiv

Article Title: EV-Elute: a universal platform for enrichment of functional surface marker-defined extracellular vesicle subpopulations

doi: 10.1101/2023.10.25.563755

Figure Lengend Snippet: A: Schematic illustrating the experimental setup of EV subpopulation uptake assays. EVs were labelled with Memglow 590 and added to co-cultures of human PBMCs and eGFP-expressing endothelial cells. After 4 hours, uptake by individual cell types was analyzed by multiplexed flow cytometry. B: Representative uptake experiment showing absolute uptake of whole EV isolates and subpopulations by co-cultures from a single PBMC donor. Uptake is expressed as the difference in mean fluorescence intensity (MFI) of Memglow 590 between EV-treated cells and PBS-treated controls (ΔMFI). Bars indicate mean ± SD from three technical replicates. C: Cell type specificity of whole EV isolates and EV subpopulations. Uptake of EVs by each cell type was expressed as a percentage of total EV uptake within experiments. Bars show mean ± SD of four experiments with different PBMC donors. No significant differences in cell type specificity between EV subpopulations were detected using one-way ANOVA with Tukey post-hoc test.

Article Snippet: For cell uptake and Cas9 delivery assays, EVs were labelled with MemGlow 590 Fluorogenic probe, whereas for single EV imaging, EVs were labelled with Memglow 560 Fluorogenic probe (Cytoskeleton, Inc).

Techniques: Expressing, Flow Cytometry, Fluorescence