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    New England Biolabs dcm c2925
    Dcm C2925, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dcm c2925/product/New England Biolabs
    Average 95 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    dcm c2925 - by Bioz Stars, 2020-05
    95/100 stars
      Buy from Supplier

    99
    Thermo Fisher c7025
    Spikes enhance interaction between apoptotic cells and phagocytes (A) Proportion of THP-1 macrophages interacting (bound and engulfed) and engulfing apoptotic A431 cells. Target cells were generated in the absence or presence of nocodazole. Shown are means (±S.E.) of triplicate samples from a single representative experiment (statistical analysis by student’s t -test). (B, C) Wide-field images of <t>CellTracker-labelled</t> apoptotic A431 cells (green) interacting with THP-1 macrophages (*). Bars = 10 μm (zoom = 5 μm).
    C7025, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 508 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c7025/product/Thermo Fisher
    Average 99 stars, based on 508 article reviews
    Price from $9.99 to $1999.99
    c7025 - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    Spikes enhance interaction between apoptotic cells and phagocytes (A) Proportion of THP-1 macrophages interacting (bound and engulfed) and engulfing apoptotic A431 cells. Target cells were generated in the absence or presence of nocodazole. Shown are means (±S.E.) of triplicate samples from a single representative experiment (statistical analysis by student’s t -test). (B, C) Wide-field images of CellTracker-labelled apoptotic A431 cells (green) interacting with THP-1 macrophages (*). Bars = 10 μm (zoom = 5 μm).

    Journal: Journal of cell science

    Article Title: A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation

    doi: 10.1242/jcs.02959

    Figure Lengend Snippet: Spikes enhance interaction between apoptotic cells and phagocytes (A) Proportion of THP-1 macrophages interacting (bound and engulfed) and engulfing apoptotic A431 cells. Target cells were generated in the absence or presence of nocodazole. Shown are means (±S.E.) of triplicate samples from a single representative experiment (statistical analysis by student’s t -test). (B, C) Wide-field images of CellTracker-labelled apoptotic A431 cells (green) interacting with THP-1 macrophages (*). Bars = 10 μm (zoom = 5 μm).

    Article Snippet: Alexa594 -annexin V, Alexa488 -phalloidin and CellTracker green (CMFDA: 10 mM) were obtained from Molecular Probes.

    Techniques: Generated

    In vitro studies to determine 3C23K mechanisms of action ( A ) Clonogenic assay: Clonogenic survival of COV434-WT and COV434-MISRII cells incubated or not (medium) with MIS (0.1 or 50 nM) or 3C23K (1 or 100 μg/ml). After 15 days of culture in the presence of MIS or 3C23K, colonies were fixed with a methanol/acetic acid solution (3:1), stained with 10% Giemsa and counted. ( B ) Smad1/5 phosphorylation: Western blot analysis of serum-deprived (for 48 hours) COV434-MISRII (left panel) or NIH-OVCAR-3 (right panel) cell extracts obtained after incubation or not with MIS l (0.1, 1, 10 or 50 nM) for 1 hour using anti-phospho-Smad1/5 (Ser463/465) (41D10) rabbit MAb. MIS induced Smad1/5 phosphorylation in MISRII-overexpressing COV434 and NIH-OVCAR3 cells at all the tested concentrations in a dose-dependent manner. ( C ) Apoptosis: After incubation with 50μg/ml MAb (as indicated) or 150 nM staurosporin (positive control) for 24 hours, COV434-MISRII cells were stained using the Annexin V-FITC Apoptosis Detection Kit (Beckman Coulter IM3614). Results of one representative experiment out of four are shown and are expressed as the percentage of cell labeled with Annexin V, propidium iodide (PI) or both. ( D and E ) ADCC: COV434-MISRII cells were incubated with human NK cells (E:T ratio = 10) purified from healthy donors’ peripheral blood (D) or human, cynomolgus monkey or mouse PBMC (E:T ratio indicated in the figure) (E) and increasing concentrations of antibody (3C23K, 32C3K-CHO or irrelevant MAb) at 37°C for 4 hours. The lysis of target cells was assessed by quantifying the release of lactate dehydrogenase (LDH) by target cells in the supernatant and calculated according to the formula: % lysis = [(ER-SR)/(100-SR)]-[(NC-SR)/(100-SR)], where ER, SR and NC represent the experimental LDH release, the spontaneous LDH release (target cells without NK cells and without antibody) and the natural cytotoxicity (target cells + NK cells without antibody), respectively. ( F ) ADPC: COV434-MISRII target cells labeled with the CMFDA dye (CellTracker ™ Green, Life Technologies) and pre-incubated with increasing concentrations of antibody (3C23K or irrelevant MAb) at room temperature for 30 minutes were mixed (10:1 E:T ratio) with macrophages derived from monocytes obtained from human or cynomolgus monkey PBMC or mouse bone marrow. Living target cells were quantified by flow cytometry after 3 (human and cynomolgus monkey macrophages) or 5 days (mouse macrophages). Results are expressed as percentages relative to the corresponding isotype control.

    Journal: Oncotarget

    Article Title: The anti-tumor efficacy of 3C23K, a glyco-engineered humanized anti-MISRII antibody, in an ovarian cancer model is mainly mediated by engagement of immune effector cells

    doi: 10.18632/oncotarget.15715

    Figure Lengend Snippet: In vitro studies to determine 3C23K mechanisms of action ( A ) Clonogenic assay: Clonogenic survival of COV434-WT and COV434-MISRII cells incubated or not (medium) with MIS (0.1 or 50 nM) or 3C23K (1 or 100 μg/ml). After 15 days of culture in the presence of MIS or 3C23K, colonies were fixed with a methanol/acetic acid solution (3:1), stained with 10% Giemsa and counted. ( B ) Smad1/5 phosphorylation: Western blot analysis of serum-deprived (for 48 hours) COV434-MISRII (left panel) or NIH-OVCAR-3 (right panel) cell extracts obtained after incubation or not with MIS l (0.1, 1, 10 or 50 nM) for 1 hour using anti-phospho-Smad1/5 (Ser463/465) (41D10) rabbit MAb. MIS induced Smad1/5 phosphorylation in MISRII-overexpressing COV434 and NIH-OVCAR3 cells at all the tested concentrations in a dose-dependent manner. ( C ) Apoptosis: After incubation with 50μg/ml MAb (as indicated) or 150 nM staurosporin (positive control) for 24 hours, COV434-MISRII cells were stained using the Annexin V-FITC Apoptosis Detection Kit (Beckman Coulter IM3614). Results of one representative experiment out of four are shown and are expressed as the percentage of cell labeled with Annexin V, propidium iodide (PI) or both. ( D and E ) ADCC: COV434-MISRII cells were incubated with human NK cells (E:T ratio = 10) purified from healthy donors’ peripheral blood (D) or human, cynomolgus monkey or mouse PBMC (E:T ratio indicated in the figure) (E) and increasing concentrations of antibody (3C23K, 32C3K-CHO or irrelevant MAb) at 37°C for 4 hours. The lysis of target cells was assessed by quantifying the release of lactate dehydrogenase (LDH) by target cells in the supernatant and calculated according to the formula: % lysis = [(ER-SR)/(100-SR)]-[(NC-SR)/(100-SR)], where ER, SR and NC represent the experimental LDH release, the spontaneous LDH release (target cells without NK cells and without antibody) and the natural cytotoxicity (target cells + NK cells without antibody), respectively. ( F ) ADPC: COV434-MISRII target cells labeled with the CMFDA dye (CellTracker ™ Green, Life Technologies) and pre-incubated with increasing concentrations of antibody (3C23K or irrelevant MAb) at room temperature for 30 minutes were mixed (10:1 E:T ratio) with macrophages derived from monocytes obtained from human or cynomolgus monkey PBMC or mouse bone marrow. Living target cells were quantified by flow cytometry after 3 (human and cynomolgus monkey macrophages) or 5 days (mouse macrophages). Results are expressed as percentages relative to the corresponding isotype control.

    Article Snippet: COV434-MISRII target cells (T) were extemporaneously labeled with the CMFDA dye (CellTracker™ Green, Life Technologies) following the manufacturer's instructions and pre-incubated with increasing concentrations of antibody (3C23K or irrelevant MAb) in RPMI/10% FBS at room temperature for 30 minutes before mixing with macrophages (effector cells: E) at a 10:1 E:T ratio.

    Techniques: In Vitro, Clonogenic Assay, Incubation, Staining, Western Blot, Positive Control, Labeling, Purification, Lysis, Derivative Assay, Flow Cytometry, Cytometry

    Mast cells are in close approximation to endothelial cells. SVEC4-10 cells co-cultured with P815 mast cells. SVEC4-10 cells were labeled with CellTracker™ Green CMFDA and P815 mast cells were labeled with CellTracker™ Red CMTPX. After 5 h of co-culture, SVEC4-10 cells (green) and P815 mast cells (red) are associated. Mast cells are in contact with endothelial cells (arrow). Inset: High magnification of the area delimited by dotted line. Image is representative of four independent experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: Mast Cells Interact with Endothelial Cells to Accelerate In Vitro Angiogenesis

    doi: 10.3390/ijms18122674

    Figure Lengend Snippet: Mast cells are in close approximation to endothelial cells. SVEC4-10 cells co-cultured with P815 mast cells. SVEC4-10 cells were labeled with CellTracker™ Green CMFDA and P815 mast cells were labeled with CellTracker™ Red CMTPX. After 5 h of co-culture, SVEC4-10 cells (green) and P815 mast cells (red) are associated. Mast cells are in contact with endothelial cells (arrow). Inset: High magnification of the area delimited by dotted line. Image is representative of four independent experiments.

    Article Snippet: CellTracker™ Cell Labelling SVEC4-10 cells were labeled with CellTracker™ Green CMFDA and P815 mast cells were labeled with CellTracker™ Red CMTPX (Molecular Probes, Thermo Fisher Scientific).

    Techniques: Cell Culture, Labeling, Co-Culture Assay

    Induction of live cell phagocytosis in cell cultures. (a) BMDMs were stimulated or not (−) with CpG DNA (0.5 μg/ml), IFN-γ (100 U/ml) and αIL-10R (1.25 μg/ml), or with CpG, IFN-γ, and αIL-10R (Triple) for 20 h, and were then co-cultured with CellTracker green dye (CMFDA)-labeled freshly-isolated live thymocytes for 2.5 h. Percentage of macrophages that contained CMFDA-positive live thymocytes were then analyzed using flow cytometry. (b) BMDMs were stimulated or not using cotreatment with CpG, IFN-γ, and αIL-10R (Triple) before co-culturing with CMFDA-labeled live thymocytes, and percentages of macrophages that performed phagocytosis were then analyzed using flow cytometry. BMDMs were also treated with or without (−) cycloheximide (CHX, 1 μg/ml) throughout the cotreatment period, or the D89E mutant of MFG-E8 (7 μg/ml) 30 min before incubation with thymocytes; * p

    Journal: EBioMedicine

    Article Title: Induction of Live Cell Phagocytosis by a Specific Combination of Inflammatory Stimuli

    doi: 10.1016/j.ebiom.2017.07.011

    Figure Lengend Snippet: Induction of live cell phagocytosis in cell cultures. (a) BMDMs were stimulated or not (−) with CpG DNA (0.5 μg/ml), IFN-γ (100 U/ml) and αIL-10R (1.25 μg/ml), or with CpG, IFN-γ, and αIL-10R (Triple) for 20 h, and were then co-cultured with CellTracker green dye (CMFDA)-labeled freshly-isolated live thymocytes for 2.5 h. Percentage of macrophages that contained CMFDA-positive live thymocytes were then analyzed using flow cytometry. (b) BMDMs were stimulated or not using cotreatment with CpG, IFN-γ, and αIL-10R (Triple) before co-culturing with CMFDA-labeled live thymocytes, and percentages of macrophages that performed phagocytosis were then analyzed using flow cytometry. BMDMs were also treated with or without (−) cycloheximide (CHX, 1 μg/ml) throughout the cotreatment period, or the D89E mutant of MFG-E8 (7 μg/ml) 30 min before incubation with thymocytes; * p

    Article Snippet: Apoptosis was induced in thymocytes using 10 μM dexamethasone treatments for 4 h, and in myeloid cells by UV irradiation at 200 J/cm2 and incubation for 2 h. For flow cytometric analyses, prey cells were washed twice with PBS and were incubated for 30 min with 1 μM CellTracker green dye (CMFDA) (Thermofisher).

    Techniques: Cell Culture, Labeling, Isolation, Flow Cytometry, Cytometry, Mutagenesis, Incubation

    V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HMEC-1 and the adhesion of PC-3 cells onto HUVECs. Confluent endothelial cells were treated with archazolid (arch) or DMSO (co) for 24 h. (A) Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and added to an HMEC-1 monolayer. The cells were allowed to adhere for 10 and 120 min. Non-adherent MDA-MB-231 cells were washed off. (B) Untreated PC-3 cells were stained with CellTracker Green CMFDA Dye and added to the HUVEC monolayer. The cells were allowed to adhere for 10, 30 and 60 min. Non-adherent PC-3 cells were washed off. (A, B) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HMEC-1 and the adhesion of PC-3 cells onto HUVECs. Confluent endothelial cells were treated with archazolid (arch) or DMSO (co) for 24 h. (A) Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and added to an HMEC-1 monolayer. The cells were allowed to adhere for 10 and 120 min. Non-adherent MDA-MB-231 cells were washed off. (B) Untreated PC-3 cells were stained with CellTracker Green CMFDA Dye and added to the HUVEC monolayer. The cells were allowed to adhere for 10, 30 and 60 min. Non-adherent PC-3 cells were washed off. (A, B) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Inhibition, Multiple Displacement Amplification, Staining, Fluorescence

    Overexpression of cathepsin B in endothelial cells attenuates both the basal and the archazolid-induced tumor cell adhesion. HUVECs were transfected with a plasmid containing human cathepsin B (catB) or the empty vector (pcDNA3.1(-)delta MCS). After 48 h cells were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. (A) The expression of cathepsin B was determined by western blot analysis. One representative blot out of three independently performed experiments is shown. Actin served as loading control. (B) Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye, added to the HUVEC monolayer and were allowed to adhere for 10 min. Non-adherent MDA-MB-231 cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO (control transfection). # p ≤ 0.05 versus 1 nM archazolid (control transfection).

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: Overexpression of cathepsin B in endothelial cells attenuates both the basal and the archazolid-induced tumor cell adhesion. HUVECs were transfected with a plasmid containing human cathepsin B (catB) or the empty vector (pcDNA3.1(-)delta MCS). After 48 h cells were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. (A) The expression of cathepsin B was determined by western blot analysis. One representative blot out of three independently performed experiments is shown. Actin served as loading control. (B) Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye, added to the HUVEC monolayer and were allowed to adhere for 10 min. Non-adherent MDA-MB-231 cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO (control transfection). # p ≤ 0.05 versus 1 nM archazolid (control transfection).

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Multiple Displacement Amplification, Labeling, Fluorescence

    Blocking integrin β1 subunits on MDA-MB-231 or PC-3 cells prevents the archazolid-mediated increase in tumor cell adhesion. Confluent HUVECs were treated either with archazolid or DMSO for 24 h. MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye. (A) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either MDA-MB-231 (n = 6) cells or HUVECs (n = 3). MDA-MB-231 cells were allowed to adhere for 10 min. (B) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either PC-3 cells (n = 5) or HUVECs (n = 3). PC-3 cells were allowed to adhere for 60 min. (A, B) Non-adherent tumor cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM. *p ≤ 0.05 versus DMSO control, # p ≤ 0.05 versus 1 nM archazolid.

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: Blocking integrin β1 subunits on MDA-MB-231 or PC-3 cells prevents the archazolid-mediated increase in tumor cell adhesion. Confluent HUVECs were treated either with archazolid or DMSO for 24 h. MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye. (A) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either MDA-MB-231 (n = 6) cells or HUVECs (n = 3). MDA-MB-231 cells were allowed to adhere for 10 min. (B) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either PC-3 cells (n = 5) or HUVECs (n = 3). PC-3 cells were allowed to adhere for 60 min. (A, B) Non-adherent tumor cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM. *p ≤ 0.05 versus DMSO control, # p ≤ 0.05 versus 1 nM archazolid.

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Blocking Assay, Multiple Displacement Amplification, Staining, Cell Adhesion Assay, Fluorescence

    V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HUVECs and decreases their transendothelial migration. (A, B) Confluent HUVECs were treated with archazolid (arch) or DMSO (co) for 24 h. Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and were added to the HUVEC monolayer. The cells were allowed to adhere for 10 min and 120 min. Non-adherent MDA-MB-231 cells were washed off. (A) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO control. (B) Microscopic images show unlabeled HUVECs and CellTracker Green CMFDA Dye-labeled MDA-MB-231 cells after 10 min of adhesion. Scale bar represents 200 μm. One representative image out of three independently performed experiments is shown. (C) HUVECs were grown on a porous filter membrane and were treated with archazolid for 24 h. Untreated CellTracker Green-labeled MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Transmigrated cells were quantified by measuring the fluorescence signal. Data are expressed as mean ± SEM (n = 5). *p ≤ 0.05 versus FCS control.

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HUVECs and decreases their transendothelial migration. (A, B) Confluent HUVECs were treated with archazolid (arch) or DMSO (co) for 24 h. Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and were added to the HUVEC monolayer. The cells were allowed to adhere for 10 min and 120 min. Non-adherent MDA-MB-231 cells were washed off. (A) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO control. (B) Microscopic images show unlabeled HUVECs and CellTracker Green CMFDA Dye-labeled MDA-MB-231 cells after 10 min of adhesion. Scale bar represents 200 μm. One representative image out of three independently performed experiments is shown. (C) HUVECs were grown on a porous filter membrane and were treated with archazolid for 24 h. Untreated CellTracker Green-labeled MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Transmigrated cells were quantified by measuring the fluorescence signal. Data are expressed as mean ± SEM (n = 5). *p ≤ 0.05 versus FCS control.

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Inhibition, Multiple Displacement Amplification, Migration, Staining, Fluorescence, Labeling

    Collagen is the major ECM component mediating MDA-MB-231 and PC-3 cell adhesion. Archazolid increases the amount of extracellular collagen on HUVECs. (A) 24-well plates were coated with 10 μg/ml collagen (col), fibronectin (fn) or laminin (ln) or were left uncoated (co). Untreated MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye and added into ECM-coated or uncoated wells. After 10 min of incubation, non-adherent cells were washed off. Adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus uncoated control. (B) A cell adhesion assay onto collagen was performed with untreated MDA-MB-231 or PC-3 cells (co) and MDA-MB-231 or PC-3 cells on which the integrin β1 subunit was blocked by a monoclonal antibody (ab). Data are expressed as mean ± SEM (n = 3). * ,# p ≤ 0.05 versus control. (C) Confluent HUVECs were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. Surface collagen (green) was detected by immunofluorescence staining of viable cells and nuclei (blue) were visualized by Hoechst 33342 staining. Scale bar represents 20 μm. One representative image out of three independently performed experiments is shown. The increase in extracellular collagen was quantified. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: Collagen is the major ECM component mediating MDA-MB-231 and PC-3 cell adhesion. Archazolid increases the amount of extracellular collagen on HUVECs. (A) 24-well plates were coated with 10 μg/ml collagen (col), fibronectin (fn) or laminin (ln) or were left uncoated (co). Untreated MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye and added into ECM-coated or uncoated wells. After 10 min of incubation, non-adherent cells were washed off. Adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus uncoated control. (B) A cell adhesion assay onto collagen was performed with untreated MDA-MB-231 or PC-3 cells (co) and MDA-MB-231 or PC-3 cells on which the integrin β1 subunit was blocked by a monoclonal antibody (ab). Data are expressed as mean ± SEM (n = 3). * ,# p ≤ 0.05 versus control. (C) Confluent HUVECs were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. Surface collagen (green) was detected by immunofluorescence staining of viable cells and nuclei (blue) were visualized by Hoechst 33342 staining. Scale bar represents 20 μm. One representative image out of three independently performed experiments is shown. The increase in extracellular collagen was quantified. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Multiple Displacement Amplification, Staining, Incubation, Fluorescence, Cell Adhesion Assay, Immunofluorescence

    Interactions between placental EVs and TLR-9 of HMEC-1 cells. Optical sections by fluorescence confocal microscopy showing HMEC-1 cells ( A ) that have been cultured with CellTracker TM Green CMFDA-labelled placental micro- ( B ) or nano- ( C ) vesicles (green). Lysosomes of HMEC-1 cells were labelled with LysoTracker® Red DND-99 (red) and the nuclei of HMEC-1 cells were counterstained with Hoechst (blue). Arrows show areas of co-localisation between placental EVs and lysosomes. Scale bar = 10 µm. Micro- and nano- vesicles collected from ID2-treated placentae were added to HMEC-1 cells with or without a TLR-9 antagonist (1 µM, antag, n = 6 placentae). As a positive control, HMEC-1 cells were treated with a TLR-9 agonist (5 µM). Surface ICAM-1 expression was measured by cell-based ELISA and normalised to that of untreated HMEC-1 cells ( D ). Statistical difference was assessed by Wilcoxin matched-pairs signed ranked tests (**p

    Journal: Scientific Reports

    Article Title: Antiphospholipid antibodies increase the levels of mitochondrial DNA in placental extracellular vesicles: Alarmin-g for preeclampsia

    doi: 10.1038/s41598-017-16448-5

    Figure Lengend Snippet: Interactions between placental EVs and TLR-9 of HMEC-1 cells. Optical sections by fluorescence confocal microscopy showing HMEC-1 cells ( A ) that have been cultured with CellTracker TM Green CMFDA-labelled placental micro- ( B ) or nano- ( C ) vesicles (green). Lysosomes of HMEC-1 cells were labelled with LysoTracker® Red DND-99 (red) and the nuclei of HMEC-1 cells were counterstained with Hoechst (blue). Arrows show areas of co-localisation between placental EVs and lysosomes. Scale bar = 10 µm. Micro- and nano- vesicles collected from ID2-treated placentae were added to HMEC-1 cells with or without a TLR-9 antagonist (1 µM, antag, n = 6 placentae). As a positive control, HMEC-1 cells were treated with a TLR-9 agonist (5 µM). Surface ICAM-1 expression was measured by cell-based ELISA and normalised to that of untreated HMEC-1 cells ( D ). Statistical difference was assessed by Wilcoxin matched-pairs signed ranked tests (**p

    Article Snippet: In some experiments, ID2 (50 μg/mL), isotype-matched control antibody (50 μg/mL), patient aPL (50 μg/mL), control IgG (50 μg/mL), or a fluorescent dye CellTracker® Green CMFDA (2 µg/mL, Invitrogen) was added into the culture medium.

    Techniques: Fluorescence, Confocal Microscopy, Cell Culture, Positive Control, Expressing, In-Cell ELISA