bead-assisted flow cytometry assay  (Immunostep)

Journal: Materials Today Bio

Article Title: The mechanistic study of injectable hydrogel loaded with BMSC-exosomes in regulating the TGF-β/MMP axis to inhibit experimental myopia model

doi: 10.1016/j.mtbio.2026.103051

Figure Lengend Snippet: Synthesis, characterization, and exosomes release profile of Exos-AlgMA hydrogels. (A) Schematic diagram of the synthesis of AlgMA. (B) The 1 H NMR spectra of Alg and AlgMA. (C) Compressive stress-strain curves and (D) corresponding compressive moduli of AlgMA and Exos-AlgMA hydrogel. (n = 3; ns: no significance). (E) SEM micrograph of the porous structure of the AlgMA hydrogel. Scale bar: 50 μm. (F) TEM image of BMSC-derived exosomes. Scale bar: 50 nm. (G) Particle size distribution of exosomes measured by DLS. (H) Cumulative release profiles of exosomes from the Exos-AlgMA hydrogel over 10 days. (I, J) Bead-assisted flow cytometry analysis of exosomal markers (CD9 and CD63). Percentages indicate CD9 + or CD63 + events within the bead gate, with unstained/blank controls used to define positive thresholds.

Article Snippet: Exosome surface markers were analyzed using a bead-assisted flow cytometry method.

Techniques: Derivative Assay, Flow Cytometry

Journal: Cells

Article Title: Regulation of MT1-MMP Activity through Its Association with ERMs

doi: 10.3390/cells9020348

Figure Lengend Snippet: Mutation of the cytoplasmic RRH563 cluster regulates MT1-MMP internalization. ( A ) Confocal microscopy analyses of MT1-MMP wt or the RRH563AAA mutant colocalization with EEA-1, CD63 or LAMP-1, markers of early-, late-endosomes and lysosomes, respectively. Pearson coefficients were calculated for all mutants stained for EEA-1, CD63 or LAMP-1 n ≥ 10 cells per condition. Data represent mean ± SEM. p values were calculated using one way analysis of variance (ANOVA) with Tukey’s or Dunn’s post-hoc multiple comparison test when appropriate. * p < 0.05, *** p < 0.001. ( B ) The internalization of MT1-MMP was evaluated using a biotinylation assay. Gels show lysates of MCF-7 cells stably expressing MT1MMP-GFP or mutant MT1MMP-RRH563AAA GFP immunoprecipitated using the anti-GFP antibody after biotinylation of the cells and internalization of the biotinylated proteins, that was allowed for 0, 15 and 30 min before treating with mercaptoethane sulfonic acid (MESNA) and lysis. Control samples correspond to cells without MESNA treatment. Blotting was sequentially performed with streptavidin-HRP (StrepHRP) and anti-GFP. The graph represents the ratio of biotinylated MT1-MMP/total MT1-MMP related to that ratio in negative control sample without MESNA treatment ± SEM in three independent experiments; data were analysed by two-way ANOVA with Bonferroni’s post-test ** p < 0.01. ( C ). Bead-assisted flow cytometry analysis of GFP-tagged MT1-MMP and RRH563AAA mutant content in extracellular vesicles (EVs), derived from MCF-7 cells transfected with either wt or mutant MT1-MMP. Bar plot depicts the mean ± SEM of three independent experiments, they were analysed by two tailed unpaired Student t -test and no differences were detected between conditions.

Article Snippet: A modification of our bead-assisted flow cytometry assay [ , ], the ExoStep kit (Immunostep), was used to quantitate MT1-MMP incorporation into EVs.

Techniques: Mutagenesis, Confocal Microscopy, Staining, Cell Surface Biotinylation Assay, Stable Transfection, Expressing, Immunoprecipitation, Lysis, Negative Control, Flow Cytometry, Derivative Assay, Transfection, Two Tailed Test

Journal: Cells

Article Title: Regulation of MT1-MMP Activity through Its Association with ERMs

doi: 10.3390/cells9020348

Figure Lengend Snippet: CD151 gene deletion does not impact on MT1-MMP subcellular localization. ( A ) Expression of tetraspanin CD151 was deleted using the CRISPR/Cas9 system in MCF-7 cells and analysed by flow cytometry to confirm the absence of the tetraspanin, after staining with LIA1/1 anti-CD151 mAb and an appropriated secondary antibody. Blue histogram corresponds to wt MCF-7 cells, black to CD151 knockout (KO) cells. Negative controls of unstained MCF-7 cells and unstained CD151 KO cells are shown in light grey and red histograms, respectively. ( B ) MCF-7 CD151 KO cells were transiently transfected with MT1-MMP mEGFP and the RRH563AAA cytoplasmic mutant. Cells were stained for the endogenous active form of ERMs (phospho-ERM in red). Colocalization analyses show a significant decrease in Pearson coefficient for the RRH563AAA mutant as compared with the wild-type form of MT1-MMP. Bars 15 µm. Graph represent the colocalization analysis between the green and the red channel. Data represent mean ± SEM of n≥10 cells for each condition. Statistical significance was determined using two tailed paired Student t -test, *** p < 0.001. ( C ) MCF-7 wt or CRISPR/Cas9 CD151 cells were transfected with mEGFP-tagged MT1-MMP constructs (wt or RRH563AAA) and plated onto 20 µg/mL collagen I-coated coverslips, fixed and labelled with anti-CD63 or anti-LAMP-1. Graphs show the colocalization analysis with CD63 wt (black) and MT1MMP-RRH563AAA mutant (grey) in MCF-7 cells and of wt (solid green) and MT1MMP-RRH563AAA mutant (light green) in CRISPR/Cas9 CD151 KO cells. The degree of colocalization was evaluated by means of the Pearson coefficient (PC) represented as the mean ± SEM of more than 15 cells of 3 independent experiments and analysed using one-way ANOVA and Dunn′s multiple comparisons test * p < 0.05, ** p < 0.01. ( D ) The different MCF-7 cell lines were lysed in 1% Bri96 and immunoprecipitated with anti-GFP polyclonal Ab or IgG sepharose. Samples were run under non-reducing conditions and blotted with anti-phospho ERM or anti-CD151 (11B1) Abs. For loading control, parallel samples were resolved under reducing conditions and blotted with anti-GFP Ab. Whole cell lysates (WCL) of MCF-7 cells and CD151 KO cells stably expressing MT1-MMP wt and MT1MMP-RRH563AAA mutant were immunoblotted for phospho-ERM. Tubulin was used as loading control. No differences were found among the different cell lines.

Article Snippet: A modification of our bead-assisted flow cytometry assay [ , ], the ExoStep kit (Immunostep), was used to quantitate MT1-MMP incorporation into EVs.

Techniques: Expressing, CRISPR, Flow Cytometry, Staining, Knock-Out, Transfection, Mutagenesis, Two Tailed Test, Construct, Immunoprecipitation, Stable Transfection

Journal: Cells

Article Title: Regulation of MT1-MMP Activity through Its Association with ERMs

doi: 10.3390/cells9020348

Figure Lengend Snippet: Connection to ERMs regulate MT1-MMP autoprocessing and activity. ( A ) Flow cytometry analysis of MT1-MMP surface expression represented as the mean fold change ± SEM of the fluorescence geometric mean comparing to MT1-MMP in three independent experiments, * p < 0.05 in a two-tailed paired Student t -test. ( B ) Western blot revealed with anti-GFP antibody was used to analyse the relative abundance of autoprocessed MT1-MMP form in total cell lysates. The ratio between full active molecule versus the inactive autoprocessed form is depicted below. Tubulin was used as loading control. ( C ) A fluorescent gelatin matrix was used as a substrate to measure MT1-MMP activity. MCF-7 cells were transiently transfected with the different mEGFP-tagged MT1-MMP constructions and plated onto 2 mg/mL gelatin for 6 h, fixed and the degraded area was quantified. Bars 100 µm. Data represent mean ± SEM of n ≥ 25 cells. Data was by Kruskal–Wallis test, * p < 0.05. ( D ) MCF-7 wt or CRISPR/Cas9 CD151 cells were transfected with mEGFP-tagged MT1-MMP constructs (wt or RRH563AAA) and plated onto 2 mg/mL fluorescent gelatin for 6 h before fixation. Degradation area was measure with Image J and normalized to the Mean Fluorescent mEGFP Intensity of each cell. Data are represented as the mean ± SEM of 100–150 cells from 3 independent experiments and by one-way ANOVA with Dunn’s post-test *** p < 0.001. ( E ) Autocatalytic cleavage reduction is also detected when the RRH563AAA mutant is transfected in CRISPR/Cas9 CD151 cells. Western blot with anti-GFP antibody was used to analyse the expression of MT1-MMP (wt or RRH563AAA) in MCF-7 CRISPR/Cas9 CD151 cells. The ratio between the active enzyme versus the processed form is calculated. Actin was used as loading control. ( F ) Graph depicts the autocatalytic activity of MT1-MMP as the ratio between the active enzyme versus the processed form obtained by western-blot. Data represent the mean ± SEM of five independent experiments analysed by one way ANOVA and Bonferroni’s multiple comparison test, * p < 0.05.

Article Snippet: A modification of our bead-assisted flow cytometry assay [ , ], the ExoStep kit (Immunostep), was used to quantitate MT1-MMP incorporation into EVs.

Techniques: Activity Assay, Flow Cytometry, Expressing, Fluorescence, Two Tailed Test, Western Blot, Transfection, CRISPR, Construct, Mutagenesis