tritc-phalloidin r415  (Thermo Fisher)

Journal: Materials Today Bio

Article Title: miR-19b-3p engineered adipose-derived stem cell exosomes attenuate acute liver failure via promoting tissue repair and microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102697

Figure Lengend Snippet: Anti-inflammation and anti-oxidation effect of ADSC-EXO derived exosomal miR-19b-3p in D-GalN/LPS-challenged macrophages. (A) Flowchart of ADSCs-derived exosomes collection and isolation. (B) Transmission electron micrograph (TEM) of ADSCs-derived exosomes (ADSC-EXO), scale bar = 200 nm (left panel), scale bar = 100 nm (right panel). (C) Size distribution, concentration and intensity of ADSC-EXO were determined using Nano-sight tracking analysis (NTA). (D) Western blot analysis for identifying exosomal protein markers in ADSC-EXO, ADSCs protein as control. (E) Confocal microscopy image showing the cellular uptake of DiO- labeled exosomes by test cells in vitro . Blue: DAPI label nuclei, Green: DiO-labeled exosomes, Red: TRITC-phalloidin labeled cytoskeleton; scale bar = 25 μm. (F) The particle concentration, protein concentration, and particle-to-protein ratio of exosomes from three other batches were measured. (G)miRNA sequencing analysis of ADSC-EXO isolated ADSCs from 2 donors' adipose tissue. (H) Rat primary macrophages (RMa-bm cell) challenged with D-GalN (70 mmol/L) and LPS (100 ng/mL) were collected and detected the exosomal miRNA respond within 48 h once 4 h using RT-PCR assay. (I) ROS level of each group in RMa-bm cells was measured using flow cytometry assay after 48 h miRNA transfection and 24 h D-GalN/LPS treatment. (J) miR-19b-3p mimic/mimic NC or inhibitor/inhibitor NC were transfected to RMa-bm cells for 48 h. Then cells were challenged with D-GalN (70 mmol/L) and LPS (100 ng/mL) for 24 h and supernatant were collected. Cytokines level of TNF-α, IL-6, IL-1α and IL-10 in supernatant were measured using ELISA assay. Statistical differences were determined using unpaired Student's t -test between each 2 cohorts (ns = no significance, ∗p < 0.05, ∗∗p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Blue: DAPI label nuclei, Green: DiO-labeled exosomes, Red: TRITC-phalloidin labeled cytoskeleton; scale bar = 25 μm. (F) The particle concentration, protein concentration, and particle-to-protein ratio of exosomes from three other batches were measured. (G)miRNA sequencing analysis of ADSC-EXO isolated ADSCs from 2 donors' adipose tissue. (H) Rat primary macrophages (RMa-bm cell) challenged with D-GalN (70 mmol/L) and LPS (100 ng/mL) were collected and detected the exosomal miRNA respond within 48 h once 4 h using RT-PCR assay. (I) ROS level of each group in RMa-bm cells was measured using flow cytometry assay after 48 h miRNA transfection and 24 h D-GalN/LPS treatment. (J) miR-19b-3p mimic/mimic NC or inhibitor/inhibitor NC were transfected to RMa-bm cells for 48 h. Then cells were challenged with D-GalN (70 mmol/L) and LPS (100 ng/mL) for 24 h and supernatant were collected.

Techniques: Derivative Assay, Isolation, Transmission Assay, Concentration Assay, Western Blot, Control, Confocal Microscopy, Labeling, In Vitro, Protein Concentration, Sequencing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 and inducing mitochondrial dysfunction

doi: 10.1038/s41467-025-61901-z

Figure Lengend Snippet: a Neonatal rat primary cardiomyocytes (NRPCs) transfected with siRNA targeting OTUD1 (siOTUD1), scrambled negative control (NC), OTUD1 plasmids (OTUD1 OE ), or empty vector (EV) for 24 h, and were subsequently stimulated with high glucose (HG; 33 mM) and palmitic acid (PA; 100 μM) for 2 or 24 h. The expressions of OTUD1, p T172 -AMPK, AMPK, p-ACC, ACC, and HK-1 were detected. b NRPCs transfected with siOTUD1 or NC were stimulated with HG, combining PA for 1 h. Flow cytometry analysis of the mitochondrial reactive oxygen species levels in NRPCs. c After 24 h of HG + PA stimulation, the ATP content was detected in NRPCs. d Western blot analysis of mitochondrial respiratory chain complexes (Grim19, SDHA, UQCRC2, MTCO1, and ATP5A) in NRPCs. e , f Analysis of oxygen consumption rate (OCR) in NRPCs treated as indicated. Oligo oligomycin, FCCP carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, Rot/AA rotenone/antimycin A. g , i Representative images of rhodamine phalloidin staining for cardiomyocyte area measurement. h , j RT-qPCR analysis of hypertrophic genes ( Nppa , Nppb , Myh7 , Acta1 ) in NRPCs with HG + PA stimulation for 24 h. k RT-qPCR analysis of hypertrophic genes in AMPKα2 knockout H9C2 cells ( gAMPKα2 ) or control ( gctrl ) cells challenged with HG and PA for 24 h. For ( g , i ), scale bar = 100 μm. Source data are provided as a file. Data are presented as mean ± SEM ( a , b , d – k : n = 3 independent experiments; c : n = 6 independent experiments); For ( c ), P values were determined by two-tailed unpaired t -test; For ( f – k ), P values were determined by one-way ANOVA followed by Tukey post hoc tests; For ( f ) (ATP Production), P values were determined by Kruskal–Wallis test with Dunn post hoc tests.

Article Snippet: Cells were then incubated with TRITC-labeled Phalloidin (CA1610, Beijing Solarbio Science & Technology Co., Ltd, Beijing, China) in the dark for 30 min at room temperature.

Techniques: Transfection, Negative Control, Plasmid Preparation, Flow Cytometry, Western Blot, Staining, Quantitative RT-PCR, Knock-Out, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Dopamine selectively regulates pediatric sclera/choroid interactions through the stimulation of exosome-associated retinoic acid

doi: 10.1101/2025.07.13.664562

Figure Lengend Snippet: (A) Representative images of paediatric scleral fibroblasts (HSF9-Posterior) in collagen gels after one day in SFM, CCM, dopamine-CCM or dopamine-CCM obtained after treatment with GW4869 (1µM) or WIN18446 (8µM). Shown are z-projections of confocal stacks of images of cells stained with FITC-phalloidin. Inset shows typical masks used for measurements. Scale bar, 100 um. (B-E) Corresponding cell shape analysis comparing the aspect ratio (B) , Feret diameter (C) , Circularity (D) and solidity (E) of scleral fibroblasts. Each datapoint representing a cell (>200), from 3 independent repeats. (F) Representative images of paediatric scleral fibroblasts (HSF9-Posterior) in collagen gels after one day in SFM, CCM, dopamine-CCM and stained for phospho-myosin (green) and F-actin (red) . (G, H) Fluorescence quantitation for phospho-myosin (G) and F-actin (H) . Represented as average integrated density/cell +/- SEM (n=3). ** P<0.01, ****P<0.0001 (One-way ANOVA and Tukey’s multiple comparisons tests).

Article Snippet: The gels were then incubated with TRITC-conjugated phalloidin (HelloBio, UK; 1:20 in TBS with 1% BSA and 1% FBS) for F-actin staining at room temperature in the dark for 30 minutes, followed by washing with TBS/BSA pH 8.0 for 30 minutes.

Techniques: Staining, Fluorescence, Quantitation Assay