tritc labelled dolichos biflorus agglutinin  (Vector Laboratories)

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: Molecules

Article Title: Anti-Inflammatory and Immunomodulatory Effects of Aqueous Extracts from Green Leaves and Rhizomes of Posidonia oceanica (L.) Delile on LPS-Stimulated RAW 264.7 Macrophages

doi: 10.3390/molecules30244685

Figure Lengend Snippet: ( A ) Representative flow cytometric profiles of FITC-dextran uptake by RAW 264.7 cells cultured under control conditions, treated with 0.1 μg/mL LPS or co-treated with LPS and 10 μg/mL GLE or 0.1 μg/mL RE. Fully processed preparations without FITC-dextran were analyzed in parallel for the control of background derived from autofluorescence (negative control). ( B ) Bar graph showing the relative mean fluorescence intensity (MFI) of control (C), LPS, GLE + LPS, or RE + LPS-treated RAW 264.7 cells. All the results were analyzed for the MFI of each condition, and the MFI of either treated cell population was divided by the MFI of the controls to normalize the data. Error bars indicate the s.e.m. of three independent measurements. One-way ANOVA followed by Holm–Sidak comparison procedure was used. * p < 0.05 and ** p < 0.001 vs. LPS. Normality test vs. control passed.

Article Snippet: To quantify the effect of the co-treatments on the bulk-phase endocytic ability of the macrophages, RAW 264.7 cells were grown in control conditions or treated with LPS with or without addition of either 10 μg/mL GLE or 0.1 μg/mL RE for 24 h. Subsequently, the cells were incubated at 37 °C for 1 h with 100 μL of fluorescein isothiocyanate (FITC)-dextran (final concentration: 1 mg/mL; MW 40000; MedChem Express) and then subjected to flow cytometric analysis to evaluate their uptake, as described by Rod-In et al. [ ].

Techniques: Cell Culture, Control, Derivative Assay, Negative Control, Fluorescence, Comparison