methyl β cyclodextrin Millipore Search Results


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  • 99
    Millipore methyl β cyclodextrin
    Effect of C6-Cer on cell proliferation. A. FRTL-5 cells were preincubated for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. B. Cell proliferation measurement using cell count. Effect of C6-Cer on cell proliferation on FRTL-5 was measured by counting the cells after preincubation for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. DMSO alone was used as control. Each value gives the amount of cells per plate. C. HeLa cells were incubated with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO for 12 h. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. D. FRTL-5 cells were exposed to 0.05 mM L-erythro-C6-Cer/CholPC or C6-dihydroCer/CholPC for 24 h after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. E. FRTL-5 cells were exposed for 48 h to 0.05 mM Chol/CholPC, Chol/DMSO, or <t>Chol/m-β-cyclodextrin</t> after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. Each value is the mean ± SEM of at least 3 independent experiments. *P
    Methyl β Cyclodextrin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cytochalasin d
    TRPM8 internalization and recruitment are affected after menthol. A , Distribution of particles follow a two-state model, where equilibrium constant values during exocytic and endocytic processes vary in the presence of menthol. B , Partial inhibition of endocytosis processes after MβCD incubation. Decay constant values (τ) indicate that menthol stimulates MβCD-dependent exocytosis by ∼6 times (blue circles) in comparison with control conditions (gray circles). Purple shaded regions on the TIRF images represent higher intensity, suggesting an increase in TRPM8-containing vesicles at PM. C , Partial inhibition of exocytosis processes induced by <t>cytoD</t> incubation. Top, Images represent overall presence of particles after cytoD incubation. Purple shaded regions represent higher intensity, suggesting a decrease in the exocytic rate. Comparison of decay constant values (τ) shows that menthol has a near twofold effect on cytoD-dependent vesicle endocytosis. n = 6.
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore filipin
    Implication of CD133 in endocytosis mainly involved the clathrin pathway. A) Consequences of CD133-specific siRNA knockdown for the effectiveness of chemical modulators of known endocytic pathways in modulating Tf uptake by non-differentiated Caco-2 cells. After treatment with either vehicle alone (control), <t>filipin,</t> chlorpromazine, DMA or MβCD added with lovastatin (MβCD), CD133 high (control siRNA) and CD133 low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to 5 µg/mL Tf-Alexa 488. Cellular internalization of Tf-Alexa 488 was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the vehicle treated control. Note the absence of effect of CD133-siRNA knockdown on the major inhibition of Tf-uptake caused by chlorpromazine. Note also the reduced up regulatory effect of cholesterol extraction in the CD133 low situation (MβCD). Data represented mean ± s.e.m. of a triplicate obtained from one representative experiment that was reproduced twice. Comparisons with control: Dunnett's test, *p
    Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore water soluble cholesterol
    Implication of CD133 in endocytosis mainly involved the clathrin pathway. A) Consequences of CD133-specific siRNA knockdown for the effectiveness of chemical modulators of known endocytic pathways in modulating Tf uptake by non-differentiated Caco-2 cells. After treatment with either vehicle alone (control), <t>filipin,</t> chlorpromazine, DMA or MβCD added with lovastatin (MβCD), CD133 high (control siRNA) and CD133 low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to 5 µg/mL Tf-Alexa 488. Cellular internalization of Tf-Alexa 488 was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the vehicle treated control. Note the absence of effect of CD133-siRNA knockdown on the major inhibition of Tf-uptake caused by chlorpromazine. Note also the reduced up regulatory effect of cholesterol extraction in the CD133 low situation (MβCD). Data represented mean ± s.e.m. of a triplicate obtained from one representative experiment that was reproduced twice. Comparisons with control: Dunnett's test, *p
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    Millipore nocodazole
    EHV-1gH S440A entry into ED cells. Cells were pretreated with genistein (A), dynasore (DYN) (B), MβCD (C), EIPA, or <t>nocodazole</t> (Noc) (D) before infection with EHV-1gH S440A (MOI = 5). At 8 to 12 h after infection, monolayers were detached and the
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    Millipore chlorpromazine
    EHV-1gH S440A entry into ED cells. Cells were pretreated with genistein (A), dynasore (DYN) (B), MβCD (C), EIPA, or <t>nocodazole</t> (Noc) (D) before infection with EHV-1gH S440A (MOI = 5). At 8 to 12 h after infection, monolayers were detached and the
    Chlorpromazine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 800 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lovastatin
    Depletion of cholesterol from the plasma membrane in living cells ( A ). Growth curves of BHK cells in the presence of (▪) and without (▴) <t>lovastatin/mevalonate.</t> ( B , C ) BHK suspension cells grown with or without lovastatin/mevalonate were
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    Millipore wortmannin
    Dynamics of constitutive membrane protein endocytosis. ( a ) Schematic outline of procedures for the quantification and encoding of cell-surface proteome endocytosis, as described in Methods. ( b ) Representative confocal microscopy images of cell-surface and internalized biotinylated proteins in HeLa cells at the indicated time points (see also, Supplementary Movie 1 ). Scale bar, 20 μm. ( c ) FACS quantification of biotinylated cell-surface, and endocytosed membrane proteome in HeLa cells following 30 min of internalization; left panel: representative histograms of non-biotinylated cells, total cell-surface biotinylation, residual cell-surface signal following reductive cleavage of biotin-protein linker with MesNa and internalized membrane proteins; right panel: quantitative analysis presented as the mean±s.d. from three independent experiments each performed in duplicates. MFI, median fluorescence intensity. ( d ) Representative confocal microscopy images of HeLa cells from experiment described in ( c ). Scale bar, 20 μm; lower right panel, 10 μm. ( e ) Time-dependent internalization in HeLa cells of cell-surface proteome presented as % of total cell-surface biotinylation at t =0, that is, without induction of endocytosis. ( f ) Confocal microscopy imaging of biotinylated proteins (magenta) and the early endosome marker EEA1 (green) in HeLa cells shows co-localization following 30 min of endocytosis. Arrows in lower right panel indicate co-localization. Shown are representative images from at least 3 independent experiments. Scale bar, 10 μm; lower right panel, 3.5 μm. ( g–k ) FACS quantification of constitutive biotinylated membrane protein endocytosis at 30 min following pre-treatment of HeLa cells with ( g ) dynamin inhibitor Dynasore for 30 min, ( h ) membrane cholesterol depletion agent methyl-β-cyclodextrin (MCD) for 30 min, ( i ) low-density lipoprotein cholesterol loading for 20 h, ( j ) macropinocytosis inhibitor <t>wortmannin</t> for 1 h and ( k ) ERK1/2 phosphorylation inhibitor UO126 for 1 h at the indicated concentrations. Data are presented as % of untreated control cells (Ctrl)±s.d. from a representative experiment. NS, not significant. * P
    Wortmannin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9936 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bafilomycin a1
    siRNA-mediated depletion of V-ATPase or prolonged inhibition with <t>Bafilomycin</t> A1 leads to accumulation of large, non-constricted CCSs at the PM (a) Efficiency of transferrin internalization after siRNA-mediated depletion of V-ATPase subunits. The cells were incubated with Alexa 488 -transferrin for 7 min, fixed, and surface transferrin receptor was stained using a secondary Alexa 647 antibody. Alexa 488 /Alexa 647 ratios from three independent experiments normalised to the NTP control are plotted. (b) CALM immunofluorescence in ATP6V0C or ATP6V1B2 knockdown cells. (c, d) EM analysis of control and V-ATPase knockdown cells (combined ATP6V0C and ATP6V1B2 SMARTpools). (c) Representative micrographs of CCSs at the PM. Arrows indicate examples of CCPs connected to the PM within the section, wedges indicate structures within 250 nm of the PM, likely to be PM-connected in a different section 30 (d) Size distribution of sectioned CCSs within 250 nm from the PM (marked with wedges in (c)). The lengths of the major and minor axis were measured. Blue lines indicate means of CCSs in control cells. (e) Size distribution of CCPs connected to the PM within the section (marked with arrows in (c)). The pit depth and neck width were measured. Grey lines indicate mean neck width.(f) Kinetics of transferrin internalization in 24 h or 10 min BafA1-treated cells. Fraction of internalized 125 I-transferrin is plotted for each time point. n = 3 independent experiments. (g) Efficiency of transferrin internalization and cell surface accumulation of transferrin receptor after pre-treatment with BafA1 for 4, 10, 16, and 24 h. The assay was performed as described in (a). n = 3 independent experiments. (h) Fold changes in the total spot intensity of CALM-labelled CCPs in a time course experiment with BafA1. ). n = 3 independent experiments. (i) EM analysis of the CCSs in cells treated with BafA1 for 24 h. (j) Size distribution of sectioned CCSs within 250 nm from the PM in BafA1-treated cells, analyzed as described for (d). (k) Size distribution of PM-connected CCPs in BafA1-treated cells, analyzed as described for (e). All error bars: ± SEM.
    Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore methyl β cyclodextrin mbcd
    Membrane cholesterol depletion differentially affects endocytosis of MPS and PS NPs. RBL cells (30,000/cm 2 ) adherent on cover-slips were pulse-labeled for 5 minutes with 10 μg of MPS or 75 μg of PS NPs or both in complete or serum-free medium as indicated. Parallel cultures were preincubated for 1 hour, with 5 mM <t>MbCD</t> in serum-free medium used to assess the involvement of cholesterol-dependent mechanisms in the uptake of NPs. After this, cells were washed and imaged under the fluorescence microscope. Representative images of two independent experiments in triplicate are shown. Abbreviations: MbCD, <t>methyl-β-cyclodextrin;</t> MPS, mesoporous silica; NPs, nanoparticles; RBL, rat basophilic leukemia; PS, polystyrene.
    Methyl β Cyclodextrin Mbcd, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dimethyl sulfoxide dmso
    The number of <t>β-III-Tubulin</t> + retinal ganglion cells (RGC) was higher after Necrostatin-1s (Nec-1s) treatment compared to vehicle in primary rat mixed retinal cultures. Treatment with varying doses of Nec-1s were performed in duplicate and performed on three independent occasions. ( A ) The experimental design of retinal culture experiments with Nec-1s treatment. Cells were plated at a 125,000 cell density in 300 μL supplemented media, after 1 day 200 μL of supplemented media containing Nec-1s was added, to make final concentrations of 0.01 to 100 pg/μL Nec-1s. ( B ) Representative images of β-III-Tubulin + RGC (white arrow heads) treated with 100 to 0.01 pg/μL of Nec-1s. ( C ) The number of β-III-Tubulin + RGC were quantified by taking 45 images per well, and displayed as mean percentages of vehicle controls. Nec-1s provided significant protection of β-III-Tubulin + RGC compared to vehicle control treatment (PBS, 10% <t>DMSO,</t> 0.9% β-methyl-cyclodextrin). In Figure 4 B, the scale bar represents 100 μm. Values are mean ± SEM.
    Dimethyl Sulfoxide Dmso, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nystatin
    The number of <t>β-III-Tubulin</t> + retinal ganglion cells (RGC) was higher after Necrostatin-1s (Nec-1s) treatment compared to vehicle in primary rat mixed retinal cultures. Treatment with varying doses of Nec-1s were performed in duplicate and performed on three independent occasions. ( A ) The experimental design of retinal culture experiments with Nec-1s treatment. Cells were plated at a 125,000 cell density in 300 μL supplemented media, after 1 day 200 μL of supplemented media containing Nec-1s was added, to make final concentrations of 0.01 to 100 pg/μL Nec-1s. ( B ) Representative images of β-III-Tubulin + RGC (white arrow heads) treated with 100 to 0.01 pg/μL of Nec-1s. ( C ) The number of β-III-Tubulin + RGC were quantified by taking 45 images per well, and displayed as mean percentages of vehicle controls. Nec-1s provided significant protection of β-III-Tubulin + RGC compared to vehicle control treatment (PBS, 10% <t>DMSO,</t> 0.9% β-methyl-cyclodextrin). In Figure 4 B, the scale bar represents 100 μm. Values are mean ± SEM.
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    Millipore amiloride
    <t>Amiloride</t> enhances APC maturation and innate cytokine secretion. pcD-S2 at 10 µg/ml with or without 1 mM of amiloride was added in cell culture for stimulation. Surface maturation markers CD40, CD80, CD83, CD86, MHC-I, MHC-II and secreted innate cytokines IL-6, TNF-α, IL-1β, IFN-γ in supernatant, were tested at day 3 on RAW264.7 (A, B, C), JAWSII (D, E), DC2.4 (F, G), peritoneal macrophage (H, I) and spleno-DC (J, K). pEGFP was injected into footpad with or without amiloride, 24 h later, surface maturation markers were analyzed on GFP+ cells from draining LN (L). Shown is one of three independent experiments with similar results, analyzed based on Cy5+ (A, D, F), F4/80+ (H), CD11c+ (J) or GFP+ (L)cells. For peritoneal macrophage and spleno-DC, n = 3. * and ** indicate significant difference between +/− amiloride.
    Amiloride, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore triton x 100
    An IgG1 anti-Cps14 MAb captures CD9-containing microvesicles. ELISA plates coated with 5 μg/ml of 44.1 IgG1 MAb specific for the Cps14 of Pn14 were incubated with BMDC supernatants collected at different times during the culture period, and the captured CD9 was detected with 1 μg/ml of biotinylated anti-CD9 MAb. (A) BMDC supernatants were tested directly (crude supernatant), after filtration through 0.22-μm filters, or once concentrated 10-fold in 100K MWCO units by ultrafiltration. (B) In a separate experiment, both the 10-fold 100K MWCO concentrated supernatant and the filtrate from this ultrafiltration were 10-fold reconcentrated in 5K MWCO ultrafiltration units. Wells coated with an IgG1 MAb of unrelated specificity were used as controls. In the same experiment, the concentrated supernatants were treated with 1% Triton X-100 (C) or 0.2% saponin (D) for 30 min at room temperature. OD 405 nm, optical density at 405 nm.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dynasore
    Fluxes of quantum dots (6.25 μg/mL apical concentration) did not decrease in the presence of 80 μM <t>dynasore</t> for 24 hours. Transmonolayer resistance of rat alveolar epithelial cell monolayers was increased by about 30% after 24 hours of exposure to dynasore compared with control. Note: *Significantly increased from control (n = 6).
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    94
    Millipore cytochrome c oxidase
    AS-IV inhibited Aβ1-42-induced <t>cytochrome</t> c release from mitochondria in SK-N-SH cells. A. A representative blots of immunoreactive bands for cytochrome c in cytosol. B. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to β-actin. C. A representative blots of immunoreactive bands for cytochrome c in mitochondria. D. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to COX IV. # P
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    Millipore simvastatin
    <t>Simvastatin</t> did not improve Ang II-induced podocyte cholesterol accumulation and apoptosis. Podocytes were pretreated with simvastatin and stimulated with Ang II. ( A ) Quantitative analysis of the fluorescence intensity in different groups, demonstrating that there was no difference in fluorescence intensity between the Ang II + simvastatin group and the Ang II group. n = 4, * p
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    Millipore brefeldin a
    Colocalization of BODIPY-SM with organelle-specific markers. CATH.a cells were cooled at 4 °C for 10 min and then pulse-labeled with BODIPY-SM (2 μM) in serum-free culture medium for 30 min at 4 °C. Cells were stained with (A) CellMask PM stain (5 μg/ml) or were chased for 30 min at 37 °C in the presence of (B) ER-Tracker Blue-White DPX (500 nM) or (C) LysoTracker Blue DND-22 (70 nM). Cells were washed and subjected to BE (B and C). (D) Cells were stained with BODIPY-Cer (2 μM; a) in the same way as described for BODIPY-SM, or pre-incubated with nocodazole (30 μM; b) or <t>brefeldin</t> A (20 μg/ml; c) for 90 min at 37 °C and then pulse-chase-labeled with BODIPY-SM as described above.
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    Millipore bsa
    Colocalization of BODIPY-SM with organelle-specific markers. CATH.a cells were cooled at 4 °C for 10 min and then pulse-labeled with BODIPY-SM (2 μM) in serum-free culture medium for 30 min at 4 °C. Cells were stained with (A) CellMask PM stain (5 μg/ml) or were chased for 30 min at 37 °C in the presence of (B) ER-Tracker Blue-White DPX (500 nM) or (C) LysoTracker Blue DND-22 (70 nM). Cells were washed and subjected to BE (B and C). (D) Cells were stained with BODIPY-Cer (2 μM; a) in the same way as described for BODIPY-SM, or pre-incubated with nocodazole (30 μM; b) or <t>brefeldin</t> A (20 μg/ml; c) for 90 min at 37 °C and then pulse-chase-labeled with BODIPY-SM as described above.
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    Millipore ammonium chloride
    Colocalization of BODIPY-SM with organelle-specific markers. CATH.a cells were cooled at 4 °C for 10 min and then pulse-labeled with BODIPY-SM (2 μM) in serum-free culture medium for 30 min at 4 °C. Cells were stained with (A) CellMask PM stain (5 μg/ml) or were chased for 30 min at 37 °C in the presence of (B) ER-Tracker Blue-White DPX (500 nM) or (C) LysoTracker Blue DND-22 (70 nM). Cells were washed and subjected to BE (B and C). (D) Cells were stained with BODIPY-Cer (2 μM; a) in the same way as described for BODIPY-SM, or pre-incubated with nocodazole (30 μM; b) or <t>brefeldin</t> A (20 μg/ml; c) for 90 min at 37 °C and then pulse-chase-labeled with BODIPY-SM as described above.
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    Image Search Results


    Effect of C6-Cer on cell proliferation. A. FRTL-5 cells were preincubated for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. B. Cell proliferation measurement using cell count. Effect of C6-Cer on cell proliferation on FRTL-5 was measured by counting the cells after preincubation for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. DMSO alone was used as control. Each value gives the amount of cells per plate. C. HeLa cells were incubated with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO for 12 h. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. D. FRTL-5 cells were exposed to 0.05 mM L-erythro-C6-Cer/CholPC or C6-dihydroCer/CholPC for 24 h after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. E. FRTL-5 cells were exposed for 48 h to 0.05 mM Chol/CholPC, Chol/DMSO, or Chol/m-β-cyclodextrin after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. Each value is the mean ± SEM of at least 3 independent experiments. *P

    Journal: PLoS ONE

    Article Title: Complexation of C6-Ceramide with Cholesteryl Phosphocholine - A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture

    doi: 10.1371/journal.pone.0061290

    Figure Lengend Snippet: Effect of C6-Cer on cell proliferation. A. FRTL-5 cells were preincubated for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. B. Cell proliferation measurement using cell count. Effect of C6-Cer on cell proliferation on FRTL-5 was measured by counting the cells after preincubation for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. DMSO alone was used as control. Each value gives the amount of cells per plate. C. HeLa cells were incubated with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO for 12 h. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. D. FRTL-5 cells were exposed to 0.05 mM L-erythro-C6-Cer/CholPC or C6-dihydroCer/CholPC for 24 h after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. E. FRTL-5 cells were exposed for 48 h to 0.05 mM Chol/CholPC, Chol/DMSO, or Chol/m-β-cyclodextrin after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. Each value is the mean ± SEM of at least 3 independent experiments. *P

    Article Snippet: Materials Cholesterol, methyl-β-cyclodextrin, and dimethyl sulfoxide (DMSO) were obtained from Sigma/Aldrich (St. Louis, MO).

    Techniques: Cell Counting, Incubation

    Proposed mechanism for human GLP-2 receptor agonist-induced internalization and postendocytotic trafficking. The GLP-2R is localized in lipid raft domains on the cell surface in the basal state. After ligand-induced GLP-2R activation, the receptor undergoes desensitization and rapid internalization into neutral lipid raft-derived endosomes, a process that can be blocked by cholesterol sequestration with filipin or cholesterol depletion with methyl-β-cyclodextrin. The receptor is then rapidly and transiently shuttled to early endosomes and then slowly recycled to the cell surface most likely via the pH-sensitive PNRC. Prevention of endosomal acidification with monensin inhibits the recycling of the GLP-2R from the PNRC after ligand-induced endocytosis. L, receptor ligand, GLP-2.

    Journal: Molecular Biology of the Cell

    Article Title: Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization

    doi: 10.1091/mbc.E03-11-0825

    Figure Lengend Snippet: Proposed mechanism for human GLP-2 receptor agonist-induced internalization and postendocytotic trafficking. The GLP-2R is localized in lipid raft domains on the cell surface in the basal state. After ligand-induced GLP-2R activation, the receptor undergoes desensitization and rapid internalization into neutral lipid raft-derived endosomes, a process that can be blocked by cholesterol sequestration with filipin or cholesterol depletion with methyl-β-cyclodextrin. The receptor is then rapidly and transiently shuttled to early endosomes and then slowly recycled to the cell surface most likely via the pH-sensitive PNRC. Prevention of endosomal acidification with monensin inhibits the recycling of the GLP-2R from the PNRC after ligand-induced endocytosis. L, receptor ligand, GLP-2.

    Article Snippet: Forskolin, 3-isobutyl-1-methylxanthine (IBMX), forskolin, anti-FLAG (M1) antibody, anti-mouse IgG-agarose, mouse IgG, 2,2′-azino-bis(3-ethylbenthiazoline-6-sulfonic acid) reagent, protease inhibitor cocktail, phosphatase inhibitor cocktail I, filipin complex, methyl-β-cyclodextrin (MβCD), and sodium monensin were all obtained from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activation Assay, Derivative Assay

    Effect of nonsterol isoprenoids and soluble cholesterol treatments on myotube index (MI) in C2C12 myoblasts affected by statins or M β CD. C2C12 myoblasts were exposed for 24, 72, or 120 h to statins or M β CD (IC 50 ) (day 1—proliferating myoblasts, day 3—differentiating myotubes, and day 5—differentiated myotubes). ATR or SIM was administered in the listed concentration at each day of differentiation: ATR: day 1—76 μ M, day 3—46 μ M, and day 5—36 μ M; SIM: day 1—87 μ M, day 3—6 μ M, and day 5—3 μ M. M β CD was added to give the final concentration: day 1—2.7 mM, day 3—1.9 mM, and day 5–1.1 mM or vehicle control (0.1% DMSO or 2% HS DMEM) without or with selected mevalonate pathway intermediate (mevalonate 100 μ M, geranylgeraniol 10 μ M, farnesol 10 μ M, Chol-PEG 1 mM, dolichol 1 μ g/mL, and ubiquinol 10 μ g/mL). Next, cells were subjected to immunofluorescence with HO33342 to counterstain nuclei (see Materials and Methods). The myotube index was determined as the ratio of the nuclear number in myotubes (C2C12 cells with three or more nuclei) to the total number of nuclei multiplied by 100%). (a) Atorvastatin (ATR, IC 50 ) did not affect myotube index (MI) at days 1 and 3. However, it significantly diminished fraction of myonuclei in myotubes at day 5. Neither of added nonsterol isoprenoids nor cholesterol treatment could influence ATR effect. (b) Simvastatin (SIM, IC 50 ) did not change MI at day 1, but it significantly lessened myotube representation at days 3 and 5. Nonsterol isoprenoids and cholesterol reversed SIM effect at day 3 but not at day 5. (c) Methyl-beta-cyclodextrin (M β CD, IC 50 ) could not effect MI at day 1, but it significantly decreased percentage of myotubes at days 3 and 5 without any effect of nonsterol isoprenoids or cholesterol. Two-way ANOVA test for MI followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time (proliferating myoblasts, differentiating myotubes, differentiated myotubes)] amounted to F (2,337) = 80.99, p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Geranylgeraniol Prevents Statin-Dependent Myotoxicity in C2C12 Muscle Cells through RAP1 GTPase Prenylation and Cytoprotective Autophagy

    doi: 10.1155/2018/6463807

    Figure Lengend Snippet: Effect of nonsterol isoprenoids and soluble cholesterol treatments on myotube index (MI) in C2C12 myoblasts affected by statins or M β CD. C2C12 myoblasts were exposed for 24, 72, or 120 h to statins or M β CD (IC 50 ) (day 1—proliferating myoblasts, day 3—differentiating myotubes, and day 5—differentiated myotubes). ATR or SIM was administered in the listed concentration at each day of differentiation: ATR: day 1—76 μ M, day 3—46 μ M, and day 5—36 μ M; SIM: day 1—87 μ M, day 3—6 μ M, and day 5—3 μ M. M β CD was added to give the final concentration: day 1—2.7 mM, day 3—1.9 mM, and day 5–1.1 mM or vehicle control (0.1% DMSO or 2% HS DMEM) without or with selected mevalonate pathway intermediate (mevalonate 100 μ M, geranylgeraniol 10 μ M, farnesol 10 μ M, Chol-PEG 1 mM, dolichol 1 μ g/mL, and ubiquinol 10 μ g/mL). Next, cells were subjected to immunofluorescence with HO33342 to counterstain nuclei (see Materials and Methods). The myotube index was determined as the ratio of the nuclear number in myotubes (C2C12 cells with three or more nuclei) to the total number of nuclei multiplied by 100%). (a) Atorvastatin (ATR, IC 50 ) did not affect myotube index (MI) at days 1 and 3. However, it significantly diminished fraction of myonuclei in myotubes at day 5. Neither of added nonsterol isoprenoids nor cholesterol treatment could influence ATR effect. (b) Simvastatin (SIM, IC 50 ) did not change MI at day 1, but it significantly lessened myotube representation at days 3 and 5. Nonsterol isoprenoids and cholesterol reversed SIM effect at day 3 but not at day 5. (c) Methyl-beta-cyclodextrin (M β CD, IC 50 ) could not effect MI at day 1, but it significantly decreased percentage of myotubes at days 3 and 5 without any effect of nonsterol isoprenoids or cholesterol. Two-way ANOVA test for MI followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time (proliferating myoblasts, differentiating myotubes, differentiated myotubes)] amounted to F (2,337) = 80.99, p

    Article Snippet: Simvastatin, (R)-mevalonic acid lithium salt, all- trans -geranylgeraniol (GGOH), farnesol (FOH), ubiquinol (UBOH), methyl-beta-cyclodextrin (M β CD), poly(ethylene-glycol 600)-cholesterol conjugate (Chol-PEG), poly(ethylene-glycol 600) (PEG), 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), and acridine orange were purchased from Sigma Aldrich, Saint Louis, MO, USA.

    Techniques: Concentration Assay, Immunofluorescence

    Cholesterol depletion induces Bsep redistribution and dysfunction Hepatocytes in sandwich culture were treated with 5 mM methyl-β-cyclodextrin to deplete membrane cholesterol. (A) Immunoblot analysis demonstrates that InsP3R2 and Bsep expression are unaffected by cholesterol depletion. β-actin was the loading control. (B) Partial redistribution of InsP3R2 and Bsep (red) from the canalicular membrane to the cytoplasm (arrows) was observed after cholesterol depletion using confocal immunofluorescence. Mrp2 (green) was used as an apical marker and TO-PRO3 (blue) was used to label nuclei. (C) Redistribution of InsP3R2 was quantified by its normalized fluorescence intensity along a 20 µm line perpendicular to the canalicular membrane. Sharper canalicular peaks are seen in controls, while broader peaks with more cytoplasmic labeling are observed in cholesterol depleted cells (p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: TYPE 2 INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR MODULATES BILE SALT EXPORT PUMP ACTIVITY IN RAT HEPATOCYTES

    doi: 10.1002/hep.24548

    Figure Lengend Snippet: Cholesterol depletion induces Bsep redistribution and dysfunction Hepatocytes in sandwich culture were treated with 5 mM methyl-β-cyclodextrin to deplete membrane cholesterol. (A) Immunoblot analysis demonstrates that InsP3R2 and Bsep expression are unaffected by cholesterol depletion. β-actin was the loading control. (B) Partial redistribution of InsP3R2 and Bsep (red) from the canalicular membrane to the cytoplasm (arrows) was observed after cholesterol depletion using confocal immunofluorescence. Mrp2 (green) was used as an apical marker and TO-PRO3 (blue) was used to label nuclei. (C) Redistribution of InsP3R2 was quantified by its normalized fluorescence intensity along a 20 µm line perpendicular to the canalicular membrane. Sharper canalicular peaks are seen in controls, while broader peaks with more cytoplasmic labeling are observed in cholesterol depleted cells (p

    Article Snippet: Methyl-beta-cyclodextrin (mβCD) also was from Sigma-Aldrich.

    Techniques: Expressing, Immunofluorescence, Marker, Fluorescence, Labeling

    The lipid raft association of DRA and DRA-ETKFminus is sensitive to cholesterol depletion. Caco-2/BBE/EGFP-DRA ( A ) and Caco-2/BBE/EGFP-DRA-ETKFminus ( B ) cells were lysed with 1% Triton X-100 and subjected to density gradient centrifugation. Top : in untreated cells EGFP-DRA and EGFP-DRA-ETKFminus float to low-density lipid raft fractions as evidenced by their cosegregation with the raft marker flotillin. Bottom : treatment with 20 mM methyl-β-cyclodextrin (MCD) at 37°C for 60 min completely shifted both EGFP-DRA and EGFP-DRA-ETKFminus out of the low-density raft fractions, whereas flotillin was only incompletely shifted. Representative data of 4 experiments.

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts

    doi: 10.1152/ajpgi.00191.2010

    Figure Lengend Snippet: The lipid raft association of DRA and DRA-ETKFminus is sensitive to cholesterol depletion. Caco-2/BBE/EGFP-DRA ( A ) and Caco-2/BBE/EGFP-DRA-ETKFminus ( B ) cells were lysed with 1% Triton X-100 and subjected to density gradient centrifugation. Top : in untreated cells EGFP-DRA and EGFP-DRA-ETKFminus float to low-density lipid raft fractions as evidenced by their cosegregation with the raft marker flotillin. Bottom : treatment with 20 mM methyl-β-cyclodextrin (MCD) at 37°C for 60 min completely shifted both EGFP-DRA and EGFP-DRA-ETKFminus out of the low-density raft fractions, whereas flotillin was only incompletely shifted. Representative data of 4 experiments.

    Article Snippet: Nigericin, methyl-β-cyclodextrin (MCD), and Triton X-100 were from Sigma-Aldrich (Steinheim, Germany).

    Techniques: Gradient Centrifugation, Marker

    TRPM8 internalization and recruitment are affected after menthol. A , Distribution of particles follow a two-state model, where equilibrium constant values during exocytic and endocytic processes vary in the presence of menthol. B , Partial inhibition of endocytosis processes after MβCD incubation. Decay constant values (τ) indicate that menthol stimulates MβCD-dependent exocytosis by ∼6 times (blue circles) in comparison with control conditions (gray circles). Purple shaded regions on the TIRF images represent higher intensity, suggesting an increase in TRPM8-containing vesicles at PM. C , Partial inhibition of exocytosis processes induced by cytoD incubation. Top, Images represent overall presence of particles after cytoD incubation. Purple shaded regions represent higher intensity, suggesting a decrease in the exocytic rate. Comparison of decay constant values (τ) shows that menthol has a near twofold effect on cytoD-dependent vesicle endocytosis. n = 6.

    Journal: The Journal of Neuroscience

    Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

    doi: 10.1523/JNEUROSCI.3820-13.2015

    Figure Lengend Snippet: TRPM8 internalization and recruitment are affected after menthol. A , Distribution of particles follow a two-state model, where equilibrium constant values during exocytic and endocytic processes vary in the presence of menthol. B , Partial inhibition of endocytosis processes after MβCD incubation. Decay constant values (τ) indicate that menthol stimulates MβCD-dependent exocytosis by ∼6 times (blue circles) in comparison with control conditions (gray circles). Purple shaded regions on the TIRF images represent higher intensity, suggesting an increase in TRPM8-containing vesicles at PM. C , Partial inhibition of exocytosis processes induced by cytoD incubation. Top, Images represent overall presence of particles after cytoD incubation. Purple shaded regions represent higher intensity, suggesting a decrease in the exocytic rate. Comparison of decay constant values (τ) shows that menthol has a near twofold effect on cytoD-dependent vesicle endocytosis. n = 6.

    Article Snippet: External bath solution contained the following (in m m ): 140 NaCl, 10 HEPES, 5 KCl, 2 MgCl, 2 CaCl, 10 glucose (all Sigma-Aldrich), pH7.4; 295 mOsm was used to perform all imaging experiments: menthol (50–500 μ m ), icilin (1 μ m ), methyl-β-cyclodextrin (MβCD, 10 μ m ), cytochalasin D (cytoD, 1 μ m ), ionomycin (iono, 1 μ m ) (all Sigma-Aldrich); and [ N -(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide] (BCTC, 1 μ m ) and thapsigargin (1 μ m ) (both Tocris Bioscience).

    Techniques: Inhibition, Incubation

    Implication of CD133 in endocytosis mainly involved the clathrin pathway. A) Consequences of CD133-specific siRNA knockdown for the effectiveness of chemical modulators of known endocytic pathways in modulating Tf uptake by non-differentiated Caco-2 cells. After treatment with either vehicle alone (control), filipin, chlorpromazine, DMA or MβCD added with lovastatin (MβCD), CD133 high (control siRNA) and CD133 low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to 5 µg/mL Tf-Alexa 488. Cellular internalization of Tf-Alexa 488 was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the vehicle treated control. Note the absence of effect of CD133-siRNA knockdown on the major inhibition of Tf-uptake caused by chlorpromazine. Note also the reduced up regulatory effect of cholesterol extraction in the CD133 low situation (MβCD). Data represented mean ± s.e.m. of a triplicate obtained from one representative experiment that was reproduced twice. Comparisons with control: Dunnett's test, *p

    Journal: PLoS ONE

    Article Title: The Importance of the Stem Cell Marker Prominin-1/CD133 in the Uptake of Transferrin and in Iron Metabolism in Human Colon Cancer Caco-2 Cells

    doi: 10.1371/journal.pone.0025515

    Figure Lengend Snippet: Implication of CD133 in endocytosis mainly involved the clathrin pathway. A) Consequences of CD133-specific siRNA knockdown for the effectiveness of chemical modulators of known endocytic pathways in modulating Tf uptake by non-differentiated Caco-2 cells. After treatment with either vehicle alone (control), filipin, chlorpromazine, DMA or MβCD added with lovastatin (MβCD), CD133 high (control siRNA) and CD133 low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to 5 µg/mL Tf-Alexa 488. Cellular internalization of Tf-Alexa 488 was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the vehicle treated control. Note the absence of effect of CD133-siRNA knockdown on the major inhibition of Tf-uptake caused by chlorpromazine. Note also the reduced up regulatory effect of cholesterol extraction in the CD133 low situation (MβCD). Data represented mean ± s.e.m. of a triplicate obtained from one representative experiment that was reproduced twice. Comparisons with control: Dunnett's test, *p

    Article Snippet: Chlorpromazine (10 µg/mL; Sigma-Aldrich) was used to inhibit clathrin-mediated transport , while filipin (1 µg/L; Sigma-Aldrich) and dimethylamiloride (DMA, 10 µM; Sigma-Aldrich) were used to inhibit caveolae-dependent endocytosis and macropinocytosis , respectively.

    Techniques: Flow Cytometry, Cytometry, Inhibition

    EHV-1gH S440A entry into ED cells. Cells were pretreated with genistein (A), dynasore (DYN) (B), MβCD (C), EIPA, or nocodazole (Noc) (D) before infection with EHV-1gH S440A (MOI = 5). At 8 to 12 h after infection, monolayers were detached and the

    Journal: Journal of Virology

    Article Title: Glycoprotein H and ?4?1 Integrins Determine the Entry Pathway of Alphaherpesviruses

    doi: 10.1128/JVI.03522-12

    Figure Lengend Snippet: EHV-1gH S440A entry into ED cells. Cells were pretreated with genistein (A), dynasore (DYN) (B), MβCD (C), EIPA, or nocodazole (Noc) (D) before infection with EHV-1gH S440A (MOI = 5). At 8 to 12 h after infection, monolayers were detached and the

    Article Snippet: The drug concentrations used were 2 μM bafilomycin A (BFLA; Sigma) dissolved in dimethyl sulfoxide (DMSO), 10 to 100 μg/ml genistein (Sigma) dissolved in DMSO, 10 μg/ml chlorpromazine (Sigma) in PBS, 5 μg/ml filipin (Sigma) in DMSO, 5 to 20 mM methyl-β-cyclodextrin (MβCD; Sigma) in PBS, 30 μM nocodazole (Sigma) in DMSO, 75 μM 5-( N -ethyl- N -isopropyl)amiloride (EIPA; Sigma) in ethanol, and 10 to 80 μM dynasore (Sigma) in DMSO.

    Techniques: Infection

    Depletion of cholesterol from the plasma membrane in living cells ( A ). Growth curves of BHK cells in the presence of (▪) and without (▴) lovastatin/mevalonate. ( B , C ) BHK suspension cells grown with or without lovastatin/mevalonate were

    Journal:

    Article Title: Heterologously Expressed GLT-1 Associates in ~200-nm Protein-Lipid Islands

    doi: 10.1529/biophysj.106.086900

    Figure Lengend Snippet: Depletion of cholesterol from the plasma membrane in living cells ( A ). Growth curves of BHK cells in the presence of (▪) and without (▴) lovastatin/mevalonate. ( B , C ) BHK suspension cells grown with or without lovastatin/mevalonate were

    Article Snippet: Lovastatin (Sigma-Aldrich) was prepared as a 20 mM stock solution as described ( ).

    Techniques:

    Dynamics of constitutive membrane protein endocytosis. ( a ) Schematic outline of procedures for the quantification and encoding of cell-surface proteome endocytosis, as described in Methods. ( b ) Representative confocal microscopy images of cell-surface and internalized biotinylated proteins in HeLa cells at the indicated time points (see also, Supplementary Movie 1 ). Scale bar, 20 μm. ( c ) FACS quantification of biotinylated cell-surface, and endocytosed membrane proteome in HeLa cells following 30 min of internalization; left panel: representative histograms of non-biotinylated cells, total cell-surface biotinylation, residual cell-surface signal following reductive cleavage of biotin-protein linker with MesNa and internalized membrane proteins; right panel: quantitative analysis presented as the mean±s.d. from three independent experiments each performed in duplicates. MFI, median fluorescence intensity. ( d ) Representative confocal microscopy images of HeLa cells from experiment described in ( c ). Scale bar, 20 μm; lower right panel, 10 μm. ( e ) Time-dependent internalization in HeLa cells of cell-surface proteome presented as % of total cell-surface biotinylation at t =0, that is, without induction of endocytosis. ( f ) Confocal microscopy imaging of biotinylated proteins (magenta) and the early endosome marker EEA1 (green) in HeLa cells shows co-localization following 30 min of endocytosis. Arrows in lower right panel indicate co-localization. Shown are representative images from at least 3 independent experiments. Scale bar, 10 μm; lower right panel, 3.5 μm. ( g–k ) FACS quantification of constitutive biotinylated membrane protein endocytosis at 30 min following pre-treatment of HeLa cells with ( g ) dynamin inhibitor Dynasore for 30 min, ( h ) membrane cholesterol depletion agent methyl-β-cyclodextrin (MCD) for 30 min, ( i ) low-density lipoprotein cholesterol loading for 20 h, ( j ) macropinocytosis inhibitor wortmannin for 1 h and ( k ) ERK1/2 phosphorylation inhibitor UO126 for 1 h at the indicated concentrations. Data are presented as % of untreated control cells (Ctrl)±s.d. from a representative experiment. NS, not significant. * P

    Journal: Nature Communications

    Article Title: Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells

    doi: 10.1038/ncomms11371

    Figure Lengend Snippet: Dynamics of constitutive membrane protein endocytosis. ( a ) Schematic outline of procedures for the quantification and encoding of cell-surface proteome endocytosis, as described in Methods. ( b ) Representative confocal microscopy images of cell-surface and internalized biotinylated proteins in HeLa cells at the indicated time points (see also, Supplementary Movie 1 ). Scale bar, 20 μm. ( c ) FACS quantification of biotinylated cell-surface, and endocytosed membrane proteome in HeLa cells following 30 min of internalization; left panel: representative histograms of non-biotinylated cells, total cell-surface biotinylation, residual cell-surface signal following reductive cleavage of biotin-protein linker with MesNa and internalized membrane proteins; right panel: quantitative analysis presented as the mean±s.d. from three independent experiments each performed in duplicates. MFI, median fluorescence intensity. ( d ) Representative confocal microscopy images of HeLa cells from experiment described in ( c ). Scale bar, 20 μm; lower right panel, 10 μm. ( e ) Time-dependent internalization in HeLa cells of cell-surface proteome presented as % of total cell-surface biotinylation at t =0, that is, without induction of endocytosis. ( f ) Confocal microscopy imaging of biotinylated proteins (magenta) and the early endosome marker EEA1 (green) in HeLa cells shows co-localization following 30 min of endocytosis. Arrows in lower right panel indicate co-localization. Shown are representative images from at least 3 independent experiments. Scale bar, 10 μm; lower right panel, 3.5 μm. ( g–k ) FACS quantification of constitutive biotinylated membrane protein endocytosis at 30 min following pre-treatment of HeLa cells with ( g ) dynamin inhibitor Dynasore for 30 min, ( h ) membrane cholesterol depletion agent methyl-β-cyclodextrin (MCD) for 30 min, ( i ) low-density lipoprotein cholesterol loading for 20 h, ( j ) macropinocytosis inhibitor wortmannin for 1 h and ( k ) ERK1/2 phosphorylation inhibitor UO126 for 1 h at the indicated concentrations. Data are presented as % of untreated control cells (Ctrl)±s.d. from a representative experiment. NS, not significant. * P

    Article Snippet: In some cases, cells were pre-treated prior to biotinylation, that is, with Dynasore (100 μM, 30 min; Sigma-Aldrich), methyl-β-cyclodextrin (5 mM, 1 h; Sigma-Aldrich), human low-density lipoproteins (100 μg ml−1 , 20 h; Intracel), U0126 (10 μM, 1 h; Selleck Chemicals) or Wortmannin (240 nM, 1 h; Sigma-Aldrich) in SF medium.

    Techniques: Confocal Microscopy, FACS, Fluorescence, Imaging, Marker

    siRNA-mediated depletion of V-ATPase or prolonged inhibition with Bafilomycin A1 leads to accumulation of large, non-constricted CCSs at the PM (a) Efficiency of transferrin internalization after siRNA-mediated depletion of V-ATPase subunits. The cells were incubated with Alexa 488 -transferrin for 7 min, fixed, and surface transferrin receptor was stained using a secondary Alexa 647 antibody. Alexa 488 /Alexa 647 ratios from three independent experiments normalised to the NTP control are plotted. (b) CALM immunofluorescence in ATP6V0C or ATP6V1B2 knockdown cells. (c, d) EM analysis of control and V-ATPase knockdown cells (combined ATP6V0C and ATP6V1B2 SMARTpools). (c) Representative micrographs of CCSs at the PM. Arrows indicate examples of CCPs connected to the PM within the section, wedges indicate structures within 250 nm of the PM, likely to be PM-connected in a different section 30 (d) Size distribution of sectioned CCSs within 250 nm from the PM (marked with wedges in (c)). The lengths of the major and minor axis were measured. Blue lines indicate means of CCSs in control cells. (e) Size distribution of CCPs connected to the PM within the section (marked with arrows in (c)). The pit depth and neck width were measured. Grey lines indicate mean neck width.(f) Kinetics of transferrin internalization in 24 h or 10 min BafA1-treated cells. Fraction of internalized 125 I-transferrin is plotted for each time point. n = 3 independent experiments. (g) Efficiency of transferrin internalization and cell surface accumulation of transferrin receptor after pre-treatment with BafA1 for 4, 10, 16, and 24 h. The assay was performed as described in (a). n = 3 independent experiments. (h) Fold changes in the total spot intensity of CALM-labelled CCPs in a time course experiment with BafA1. ). n = 3 independent experiments. (i) EM analysis of the CCSs in cells treated with BafA1 for 24 h. (j) Size distribution of sectioned CCSs within 250 nm from the PM in BafA1-treated cells, analyzed as described for (d). (k) Size distribution of PM-connected CCPs in BafA1-treated cells, analyzed as described for (e). All error bars: ± SEM.

    Journal: Nature cell biology

    Article Title: A Human Genome-wide Screen for Regulators of Clathrin-coated Vesicle Formation Reveals an Unexpected Role for the V-ATPase

    doi: 10.1038/ncb2652

    Figure Lengend Snippet: siRNA-mediated depletion of V-ATPase or prolonged inhibition with Bafilomycin A1 leads to accumulation of large, non-constricted CCSs at the PM (a) Efficiency of transferrin internalization after siRNA-mediated depletion of V-ATPase subunits. The cells were incubated with Alexa 488 -transferrin for 7 min, fixed, and surface transferrin receptor was stained using a secondary Alexa 647 antibody. Alexa 488 /Alexa 647 ratios from three independent experiments normalised to the NTP control are plotted. (b) CALM immunofluorescence in ATP6V0C or ATP6V1B2 knockdown cells. (c, d) EM analysis of control and V-ATPase knockdown cells (combined ATP6V0C and ATP6V1B2 SMARTpools). (c) Representative micrographs of CCSs at the PM. Arrows indicate examples of CCPs connected to the PM within the section, wedges indicate structures within 250 nm of the PM, likely to be PM-connected in a different section 30 (d) Size distribution of sectioned CCSs within 250 nm from the PM (marked with wedges in (c)). The lengths of the major and minor axis were measured. Blue lines indicate means of CCSs in control cells. (e) Size distribution of CCPs connected to the PM within the section (marked with arrows in (c)). The pit depth and neck width were measured. Grey lines indicate mean neck width.(f) Kinetics of transferrin internalization in 24 h or 10 min BafA1-treated cells. Fraction of internalized 125 I-transferrin is plotted for each time point. n = 3 independent experiments. (g) Efficiency of transferrin internalization and cell surface accumulation of transferrin receptor after pre-treatment with BafA1 for 4, 10, 16, and 24 h. The assay was performed as described in (a). n = 3 independent experiments. (h) Fold changes in the total spot intensity of CALM-labelled CCPs in a time course experiment with BafA1. ). n = 3 independent experiments. (i) EM analysis of the CCSs in cells treated with BafA1 for 24 h. (j) Size distribution of sectioned CCSs within 250 nm from the PM in BafA1-treated cells, analyzed as described for (d). (k) Size distribution of PM-connected CCPs in BafA1-treated cells, analyzed as described for (e). All error bars: ± SEM.

    Article Snippet: Bafilomycin A1 (Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 100 nM; corresponding volume of DMSO (1:1000, Sigma) was added as control.

    Techniques: Inhibition, Incubation, Staining, Immunofluorescence

    Membrane cholesterol depletion differentially affects endocytosis of MPS and PS NPs. RBL cells (30,000/cm 2 ) adherent on cover-slips were pulse-labeled for 5 minutes with 10 μg of MPS or 75 μg of PS NPs or both in complete or serum-free medium as indicated. Parallel cultures were preincubated for 1 hour, with 5 mM MbCD in serum-free medium used to assess the involvement of cholesterol-dependent mechanisms in the uptake of NPs. After this, cells were washed and imaged under the fluorescence microscope. Representative images of two independent experiments in triplicate are shown. Abbreviations: MbCD, methyl-β-cyclodextrin; MPS, mesoporous silica; NPs, nanoparticles; RBL, rat basophilic leukemia; PS, polystyrene.

    Journal: International Journal of Nanomedicine

    Article Title: Labeling and exocytosis of secretory compartments in RBL mastocytes by polystyrene and mesoporous silica nanoparticles

    doi: 10.2147/IJN.S29034

    Figure Lengend Snippet: Membrane cholesterol depletion differentially affects endocytosis of MPS and PS NPs. RBL cells (30,000/cm 2 ) adherent on cover-slips were pulse-labeled for 5 minutes with 10 μg of MPS or 75 μg of PS NPs or both in complete or serum-free medium as indicated. Parallel cultures were preincubated for 1 hour, with 5 mM MbCD in serum-free medium used to assess the involvement of cholesterol-dependent mechanisms in the uptake of NPs. After this, cells were washed and imaged under the fluorescence microscope. Representative images of two independent experiments in triplicate are shown. Abbreviations: MbCD, methyl-β-cyclodextrin; MPS, mesoporous silica; NPs, nanoparticles; RBL, rat basophilic leukemia; PS, polystyrene.

    Article Snippet: Methyl-β-cyclodextrin (MbCD) (cod C4805; Sigma-Aldrich) was used at 5 mM final concentration.

    Techniques: Labeling, Fluorescence, Microscopy

    The number of β-III-Tubulin + retinal ganglion cells (RGC) was higher after Necrostatin-1s (Nec-1s) treatment compared to vehicle in primary rat mixed retinal cultures. Treatment with varying doses of Nec-1s were performed in duplicate and performed on three independent occasions. ( A ) The experimental design of retinal culture experiments with Nec-1s treatment. Cells were plated at a 125,000 cell density in 300 μL supplemented media, after 1 day 200 μL of supplemented media containing Nec-1s was added, to make final concentrations of 0.01 to 100 pg/μL Nec-1s. ( B ) Representative images of β-III-Tubulin + RGC (white arrow heads) treated with 100 to 0.01 pg/μL of Nec-1s. ( C ) The number of β-III-Tubulin + RGC were quantified by taking 45 images per well, and displayed as mean percentages of vehicle controls. Nec-1s provided significant protection of β-III-Tubulin + RGC compared to vehicle control treatment (PBS, 10% DMSO, 0.9% β-methyl-cyclodextrin). In Figure 4 B, the scale bar represents 100 μm. Values are mean ± SEM.

    Journal: Cells

    Article Title: Retinal Ganglion Cells Die by Necroptotic Mechanisms in a Site-Specific Manner in a Rat Blunt Ocular Injury Model

    doi: 10.3390/cells8121517

    Figure Lengend Snippet: The number of β-III-Tubulin + retinal ganglion cells (RGC) was higher after Necrostatin-1s (Nec-1s) treatment compared to vehicle in primary rat mixed retinal cultures. Treatment with varying doses of Nec-1s were performed in duplicate and performed on three independent occasions. ( A ) The experimental design of retinal culture experiments with Nec-1s treatment. Cells were plated at a 125,000 cell density in 300 μL supplemented media, after 1 day 200 μL of supplemented media containing Nec-1s was added, to make final concentrations of 0.01 to 100 pg/μL Nec-1s. ( B ) Representative images of β-III-Tubulin + RGC (white arrow heads) treated with 100 to 0.01 pg/μL of Nec-1s. ( C ) The number of β-III-Tubulin + RGC were quantified by taking 45 images per well, and displayed as mean percentages of vehicle controls. Nec-1s provided significant protection of β-III-Tubulin + RGC compared to vehicle control treatment (PBS, 10% DMSO, 0.9% β-methyl-cyclodextrin). In Figure 4 B, the scale bar represents 100 μm. Values are mean ± SEM.

    Article Snippet: Intravitreal Injection of Nec-1s Nec-1s (Bio Vision Incorporated, CA, USA) was reconstituted at 3.6 mM in 10% dimethyl sulfoxide (DMSO) and 0.9% methyl-β-cyclodextrin (Sigma) in sterile phosphate buffered saline (PBS).

    Techniques:

    Amiloride enhances APC maturation and innate cytokine secretion. pcD-S2 at 10 µg/ml with or without 1 mM of amiloride was added in cell culture for stimulation. Surface maturation markers CD40, CD80, CD83, CD86, MHC-I, MHC-II and secreted innate cytokines IL-6, TNF-α, IL-1β, IFN-γ in supernatant, were tested at day 3 on RAW264.7 (A, B, C), JAWSII (D, E), DC2.4 (F, G), peritoneal macrophage (H, I) and spleno-DC (J, K). pEGFP was injected into footpad with or without amiloride, 24 h later, surface maturation markers were analyzed on GFP+ cells from draining LN (L). Shown is one of three independent experiments with similar results, analyzed based on Cy5+ (A, D, F), F4/80+ (H), CD11c+ (J) or GFP+ (L)cells. For peritoneal macrophage and spleno-DC, n = 3. * and ** indicate significant difference between +/− amiloride.

    Journal: PLoS ONE

    Article Title: Amiloride Enhances Antigen Specific CTL by Faciliting HBV DNA Vaccine Entry into Cells

    doi: 10.1371/journal.pone.0033015

    Figure Lengend Snippet: Amiloride enhances APC maturation and innate cytokine secretion. pcD-S2 at 10 µg/ml with or without 1 mM of amiloride was added in cell culture for stimulation. Surface maturation markers CD40, CD80, CD83, CD86, MHC-I, MHC-II and secreted innate cytokines IL-6, TNF-α, IL-1β, IFN-γ in supernatant, were tested at day 3 on RAW264.7 (A, B, C), JAWSII (D, E), DC2.4 (F, G), peritoneal macrophage (H, I) and spleno-DC (J, K). pEGFP was injected into footpad with or without amiloride, 24 h later, surface maturation markers were analyzed on GFP+ cells from draining LN (L). Shown is one of three independent experiments with similar results, analyzed based on Cy5+ (A, D, F), F4/80+ (H), CD11c+ (J) or GFP+ (L)cells. For peritoneal macrophage and spleno-DC, n = 3. * and ** indicate significant difference between +/− amiloride.

    Article Snippet: After medium was removed, cells were treated with amiloride, MβCD (5 mM, Sigma) or Fillipin (10 µg/ml, Sigma) at 37°C for 1 h. The cells were treated with LPS (10 ng/ml, Sigma) or 10 µg/ml DNA in DMEM at 37°C for 0.5 h and washed 3 times.

    Techniques: Cell Culture, Injection

    Amiloride enhances adaptive immunity against HBV S2. Naïve C57 mice were immunized s.c. with pcD-S2 at various concentrations with or without amiloride in the hind footpad four times using the immunization scheme as shown (A). Seven days after the last immunization, animals (n = 3) were used to test anti-S2 IgG antibody titer (B) and delayed hypersensitivity (DTH) response after re-stimulation with 1 µg sAg s.c. in a hind footpad for 24 h (C). PBS was added as negative control. *, statistical significance among all groups. Another 3 animals were used to test HBV S208–215 specific lysis in vitro (D) and in vivo (E). Hepatocytes from HBV Alb1 trangenic mice were used as targets mixed with effectors from the immunized mice, or transferred into the immunized mice before the analysis of specific lysis in vitro (F) and in vivo (G). *, statistical significance between +/− amiloride.

    Journal: PLoS ONE

    Article Title: Amiloride Enhances Antigen Specific CTL by Faciliting HBV DNA Vaccine Entry into Cells

    doi: 10.1371/journal.pone.0033015

    Figure Lengend Snippet: Amiloride enhances adaptive immunity against HBV S2. Naïve C57 mice were immunized s.c. with pcD-S2 at various concentrations with or without amiloride in the hind footpad four times using the immunization scheme as shown (A). Seven days after the last immunization, animals (n = 3) were used to test anti-S2 IgG antibody titer (B) and delayed hypersensitivity (DTH) response after re-stimulation with 1 µg sAg s.c. in a hind footpad for 24 h (C). PBS was added as negative control. *, statistical significance among all groups. Another 3 animals were used to test HBV S208–215 specific lysis in vitro (D) and in vivo (E). Hepatocytes from HBV Alb1 trangenic mice were used as targets mixed with effectors from the immunized mice, or transferred into the immunized mice before the analysis of specific lysis in vitro (F) and in vivo (G). *, statistical significance between +/− amiloride.

    Article Snippet: After medium was removed, cells were treated with amiloride, MβCD (5 mM, Sigma) or Fillipin (10 µg/ml, Sigma) at 37°C for 1 h. The cells were treated with LPS (10 ng/ml, Sigma) or 10 µg/ml DNA in DMEM at 37°C for 0.5 h and washed 3 times.

    Techniques: Mouse Assay, Negative Control, Lysis, In Vitro, In Vivo

    Amiloride facilitates plasmid entry in vitro . Cy5-pEGFP entry into cell lines with or without 1 mM amiloride was analyzed after 2 h and is shown as the percentage of cells that were Cy5 + . Expression of GFP was analyzed at day 3 and is shown as percentage of EGFP + Cy5 + , on RAW264.7 (A, B, C), JAWSII (D, E), and DC2.4 (F, G). Data represent one of three independent experiments.

    Journal: PLoS ONE

    Article Title: Amiloride Enhances Antigen Specific CTL by Faciliting HBV DNA Vaccine Entry into Cells

    doi: 10.1371/journal.pone.0033015

    Figure Lengend Snippet: Amiloride facilitates plasmid entry in vitro . Cy5-pEGFP entry into cell lines with or without 1 mM amiloride was analyzed after 2 h and is shown as the percentage of cells that were Cy5 + . Expression of GFP was analyzed at day 3 and is shown as percentage of EGFP + Cy5 + , on RAW264.7 (A, B, C), JAWSII (D, E), and DC2.4 (F, G). Data represent one of three independent experiments.

    Article Snippet: After medium was removed, cells were treated with amiloride, MβCD (5 mM, Sigma) or Fillipin (10 µg/ml, Sigma) at 37°C for 1 h. The cells were treated with LPS (10 ng/ml, Sigma) or 10 µg/ml DNA in DMEM at 37°C for 0.5 h and washed 3 times.

    Techniques: Plasmid Preparation, In Vitro, Expressing

    Amiloride increases the frequency of triple positive CD8 T cells. Splenocytes from mice immunized with pcD-S2 with or without amiloride (n = 3) were re-stimulated in vitro with 10 µg/ml S208–215 for 12 h (A-C), or 10 µg/ml HBsAg for 24 h (D), then cytokine secretion was blocked by monensin for 6 h. PMA or Ionomycin stimulating splenocyte of pcD-S2 immunized mice was added as positive controls. Cells stained with anti-CD3 and anti-CD8 were gated and then used for intracellular staining with single or multiple fluorescent-labeled antibodies. A shows the proportion of responsive cells in the total CD8 + T cells with or without amiloride treatment. These responsive cells were designated as either IFN-γ + , perforin + , or granzymeB + cells. B shows the cytokine expression pattern in the responsive CD8 T cells with or without amiloride treatment. C shows the dose dependent effects of amiloride on the frequency of (IFN-γ + perforin + granzymeB + ) triple positive cells. D shows the change in frequency of triple positive cells in response to HBsAg re-stimulation. The change in frequency of triple positive cells after co-culture with peritoneal macrophages followed by re-stimulation with S208–215 (E), or with spleno-DC (F) are also shown. Data represent three independent experiments with similar results.

    Journal: PLoS ONE

    Article Title: Amiloride Enhances Antigen Specific CTL by Faciliting HBV DNA Vaccine Entry into Cells

    doi: 10.1371/journal.pone.0033015

    Figure Lengend Snippet: Amiloride increases the frequency of triple positive CD8 T cells. Splenocytes from mice immunized with pcD-S2 with or without amiloride (n = 3) were re-stimulated in vitro with 10 µg/ml S208–215 for 12 h (A-C), or 10 µg/ml HBsAg for 24 h (D), then cytokine secretion was blocked by monensin for 6 h. PMA or Ionomycin stimulating splenocyte of pcD-S2 immunized mice was added as positive controls. Cells stained with anti-CD3 and anti-CD8 were gated and then used for intracellular staining with single or multiple fluorescent-labeled antibodies. A shows the proportion of responsive cells in the total CD8 + T cells with or without amiloride treatment. These responsive cells were designated as either IFN-γ + , perforin + , or granzymeB + cells. B shows the cytokine expression pattern in the responsive CD8 T cells with or without amiloride treatment. C shows the dose dependent effects of amiloride on the frequency of (IFN-γ + perforin + granzymeB + ) triple positive cells. D shows the change in frequency of triple positive cells in response to HBsAg re-stimulation. The change in frequency of triple positive cells after co-culture with peritoneal macrophages followed by re-stimulation with S208–215 (E), or with spleno-DC (F) are also shown. Data represent three independent experiments with similar results.

    Article Snippet: After medium was removed, cells were treated with amiloride, MβCD (5 mM, Sigma) or Fillipin (10 µg/ml, Sigma) at 37°C for 1 h. The cells were treated with LPS (10 ng/ml, Sigma) or 10 µg/ml DNA in DMEM at 37°C for 0.5 h and washed 3 times.

    Techniques: Mouse Assay, In Vitro, Staining, Labeling, Expressing, Co-Culture Assay

    Amiloride accelerates plasmid entry in vivo . Naïve C57 mice (n = 3) were immunized with Cy5-pEGFP s.c. in hind footpad with (right side) or without (left side) amiloride. Lymph node cells were collected after 4 h (Cy5 + cells) and after 24 h (GFP + cells) and examined for proportion (B, D) and subtype (C, E) of stained cells from both draining and control lymph nodes. * in B and D, statistical significance between control and draining lymph nodes in all treated groups.

    Journal: PLoS ONE

    Article Title: Amiloride Enhances Antigen Specific CTL by Faciliting HBV DNA Vaccine Entry into Cells

    doi: 10.1371/journal.pone.0033015

    Figure Lengend Snippet: Amiloride accelerates plasmid entry in vivo . Naïve C57 mice (n = 3) were immunized with Cy5-pEGFP s.c. in hind footpad with (right side) or without (left side) amiloride. Lymph node cells were collected after 4 h (Cy5 + cells) and after 24 h (GFP + cells) and examined for proportion (B, D) and subtype (C, E) of stained cells from both draining and control lymph nodes. * in B and D, statistical significance between control and draining lymph nodes in all treated groups.

    Article Snippet: After medium was removed, cells were treated with amiloride, MβCD (5 mM, Sigma) or Fillipin (10 µg/ml, Sigma) at 37°C for 1 h. The cells were treated with LPS (10 ng/ml, Sigma) or 10 µg/ml DNA in DMEM at 37°C for 0.5 h and washed 3 times.

    Techniques: Plasmid Preparation, In Vivo, Mouse Assay, Staining

    An IgG1 anti-Cps14 MAb captures CD9-containing microvesicles. ELISA plates coated with 5 μg/ml of 44.1 IgG1 MAb specific for the Cps14 of Pn14 were incubated with BMDC supernatants collected at different times during the culture period, and the captured CD9 was detected with 1 μg/ml of biotinylated anti-CD9 MAb. (A) BMDC supernatants were tested directly (crude supernatant), after filtration through 0.22-μm filters, or once concentrated 10-fold in 100K MWCO units by ultrafiltration. (B) In a separate experiment, both the 10-fold 100K MWCO concentrated supernatant and the filtrate from this ultrafiltration were 10-fold reconcentrated in 5K MWCO ultrafiltration units. Wells coated with an IgG1 MAb of unrelated specificity were used as controls. In the same experiment, the concentrated supernatants were treated with 1% Triton X-100 (C) or 0.2% saponin (D) for 30 min at room temperature. OD 405 nm, optical density at 405 nm.

    Journal: Infection and Immunity

    Article Title: Dendritic Cell-Derived Exosomes Express a Streptococcus pneumoniae Capsular Polysaccharide Type 14 Cross-Reactive Antigen That Induces Protective Immunoglobulin Responses against Pneumococcal Infection in Mice ▿

    doi: 10.1128/IAI.01217-06

    Figure Lengend Snippet: An IgG1 anti-Cps14 MAb captures CD9-containing microvesicles. ELISA plates coated with 5 μg/ml of 44.1 IgG1 MAb specific for the Cps14 of Pn14 were incubated with BMDC supernatants collected at different times during the culture period, and the captured CD9 was detected with 1 μg/ml of biotinylated anti-CD9 MAb. (A) BMDC supernatants were tested directly (crude supernatant), after filtration through 0.22-μm filters, or once concentrated 10-fold in 100K MWCO units by ultrafiltration. (B) In a separate experiment, both the 10-fold 100K MWCO concentrated supernatant and the filtrate from this ultrafiltration were 10-fold reconcentrated in 5K MWCO ultrafiltration units. Wells coated with an IgG1 MAb of unrelated specificity were used as controls. In the same experiment, the concentrated supernatants were treated with 1% Triton X-100 (C) or 0.2% saponin (D) for 30 min at room temperature. OD 405 nm, optical density at 405 nm.

    Article Snippet: Concentrated supernatants were treated for 30 min at room temperature with 1% Triton X-100 or 0.0025 to 0.5% saponin from quillaja bark (Sigma, Saint Louis, MO), or at 37°C with 0.05 to 50 mM methyl-β-cyclodextrin (MbCD; Sigma).

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Filtration

    BMDC constitutively release CD9-containing microvesicles. (A) BMDC supernatants collected at different times during the culture period were filtered through 0.22-μm filters and treated with 0.2% saponin or 1% Triton X-100 for 30 min at room temperature. Microvesical CD9 content was estimated by sandwich-capture ELISA using the same anti-CD9 MAb for capture and detection. Results are expressed as μg/ml of protein exosomes using a preparation of purified BMDC-derived exosomes as a standard. Data represent the arithmetic means ± SEM of three wells tested separately. (B) CD9 in BMDC supernatants was captured using anti-CD9 (“anti-CD9 captured”) or anti-CD81 MAbs (“anti-CD81 captured”). In both cases, captured microvesicles were detected with biotinylated anti-CD9 MAb. (C) Culture supernatants filtered through 0.22-μm filters were concentrated 10-fold by ultrafiltration through 100K MWCO units, and the eluted solution was 10-fold concentrated in 5K MWCO units. Both concentrated samples were diluted to the initial volume to obtain a direct comparison with the nonconcentrated culture supernatant. As a reference, crude supernatant (unfiltered through 0.22-μm filters) was included in the assay. Data displayed in each graph are from three separate experiments. OD 405 nm, optical density at 405 nm.

    Journal: Infection and Immunity

    Article Title: Dendritic Cell-Derived Exosomes Express a Streptococcus pneumoniae Capsular Polysaccharide Type 14 Cross-Reactive Antigen That Induces Protective Immunoglobulin Responses against Pneumococcal Infection in Mice ▿

    doi: 10.1128/IAI.01217-06

    Figure Lengend Snippet: BMDC constitutively release CD9-containing microvesicles. (A) BMDC supernatants collected at different times during the culture period were filtered through 0.22-μm filters and treated with 0.2% saponin or 1% Triton X-100 for 30 min at room temperature. Microvesical CD9 content was estimated by sandwich-capture ELISA using the same anti-CD9 MAb for capture and detection. Results are expressed as μg/ml of protein exosomes using a preparation of purified BMDC-derived exosomes as a standard. Data represent the arithmetic means ± SEM of three wells tested separately. (B) CD9 in BMDC supernatants was captured using anti-CD9 (“anti-CD9 captured”) or anti-CD81 MAbs (“anti-CD81 captured”). In both cases, captured microvesicles were detected with biotinylated anti-CD9 MAb. (C) Culture supernatants filtered through 0.22-μm filters were concentrated 10-fold by ultrafiltration through 100K MWCO units, and the eluted solution was 10-fold concentrated in 5K MWCO units. Both concentrated samples were diluted to the initial volume to obtain a direct comparison with the nonconcentrated culture supernatant. As a reference, crude supernatant (unfiltered through 0.22-μm filters) was included in the assay. Data displayed in each graph are from three separate experiments. OD 405 nm, optical density at 405 nm.

    Article Snippet: Concentrated supernatants were treated for 30 min at room temperature with 1% Triton X-100 or 0.0025 to 0.5% saponin from quillaja bark (Sigma, Saint Louis, MO), or at 37°C with 0.05 to 50 mM methyl-β-cyclodextrin (MbCD; Sigma).

    Techniques: Enzyme-linked Immunosorbent Assay, Purification, Derivative Assay

    Fluxes of quantum dots (6.25 μg/mL apical concentration) did not decrease in the presence of 80 μM dynasore for 24 hours. Transmonolayer resistance of rat alveolar epithelial cell monolayers was increased by about 30% after 24 hours of exposure to dynasore compared with control. Note: *Significantly increased from control (n = 6).

    Journal: International Journal of Nanomedicine

    Article Title: Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers

    doi: 10.2147/IJN.S26051

    Figure Lengend Snippet: Fluxes of quantum dots (6.25 μg/mL apical concentration) did not decrease in the presence of 80 μM dynasore for 24 hours. Transmonolayer resistance of rat alveolar epithelial cell monolayers was increased by about 30% after 24 hours of exposure to dynasore compared with control. Note: *Significantly increased from control (n = 6).

    Article Snippet: Briefly, RAECM were pre-treated for 30 minutes with methyl-β-cyclodextrin (200 μM, Sigma), chlorpromazine (28 μM, Sigma), or dynasore (80 μM, Sigma) in both apical and basolateral fluids.

    Techniques: Concentration Assay

    AS-IV inhibited Aβ1-42-induced cytochrome c release from mitochondria in SK-N-SH cells. A. A representative blots of immunoreactive bands for cytochrome c in cytosol. B. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to β-actin. C. A representative blots of immunoreactive bands for cytochrome c in mitochondria. D. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to COX IV. # P

    Journal: PLoS ONE

    Article Title: Protective Effects of Astragaloside IV against Amyloid Beta1-42 Neurotoxicity by Inhibiting the Mitochondrial Permeability Transition Pore Opening

    doi: 10.1371/journal.pone.0098866

    Figure Lengend Snippet: AS-IV inhibited Aβ1-42-induced cytochrome c release from mitochondria in SK-N-SH cells. A. A representative blots of immunoreactive bands for cytochrome c in cytosol. B. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to β-actin. C. A representative blots of immunoreactive bands for cytochrome c in mitochondria. D. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to COX IV. # P

    Article Snippet: Detection of the activity of cytochrome c oxidase (CcO activity) Cytochrome c oxidase activity was measured by using the cytochrome c oxidase kit (Sigma).

    Techniques: Expressing

    Simvastatin did not improve Ang II-induced podocyte cholesterol accumulation and apoptosis. Podocytes were pretreated with simvastatin and stimulated with Ang II. ( A ) Quantitative analysis of the fluorescence intensity in different groups, demonstrating that there was no difference in fluorescence intensity between the Ang II + simvastatin group and the Ang II group. n = 4, * p

    Journal: Scientific Reports

    Article Title: Angiotensin II induces cholesterol accumulation and injury in podocytes

    doi: 10.1038/s41598-017-09733-w

    Figure Lengend Snippet: Simvastatin did not improve Ang II-induced podocyte cholesterol accumulation and apoptosis. Podocytes were pretreated with simvastatin and stimulated with Ang II. ( A ) Quantitative analysis of the fluorescence intensity in different groups, demonstrating that there was no difference in fluorescence intensity between the Ang II + simvastatin group and the Ang II group. n = 4, * p

    Article Snippet: Then, the cells were stimulated with 10−9 –10−5 M Ang II (Enzo Life Science, Netherlands) for 24 h. For losartan treatment, serum-starved podocytes were treated with 10−5 M losartan for 24 h. For CD experiments, serum-starved podocytes were pretreated with 5 mM methyl-β-cyclodextrin (Sigma-Aldrich, USA) for 1 h. For statin experiments, serum-starved podocytes were pretreated for 1 h with 1 μM simvastatin (Sigma-Aldrich, USA).

    Techniques: Fluorescence

    Colocalization of BODIPY-SM with organelle-specific markers. CATH.a cells were cooled at 4 °C for 10 min and then pulse-labeled with BODIPY-SM (2 μM) in serum-free culture medium for 30 min at 4 °C. Cells were stained with (A) CellMask PM stain (5 μg/ml) or were chased for 30 min at 37 °C in the presence of (B) ER-Tracker Blue-White DPX (500 nM) or (C) LysoTracker Blue DND-22 (70 nM). Cells were washed and subjected to BE (B and C). (D) Cells were stained with BODIPY-Cer (2 μM; a) in the same way as described for BODIPY-SM, or pre-incubated with nocodazole (30 μM; b) or brefeldin A (20 μg/ml; c) for 90 min at 37 °C and then pulse-chase-labeled with BODIPY-SM as described above.

    Journal: Biochimica et Biophysica Acta

    Article Title: Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons

    doi: 10.1016/j.bbalip.2013.08.007

    Figure Lengend Snippet: Colocalization of BODIPY-SM with organelle-specific markers. CATH.a cells were cooled at 4 °C for 10 min and then pulse-labeled with BODIPY-SM (2 μM) in serum-free culture medium for 30 min at 4 °C. Cells were stained with (A) CellMask PM stain (5 μg/ml) or were chased for 30 min at 37 °C in the presence of (B) ER-Tracker Blue-White DPX (500 nM) or (C) LysoTracker Blue DND-22 (70 nM). Cells were washed and subjected to BE (B and C). (D) Cells were stained with BODIPY-Cer (2 μM; a) in the same way as described for BODIPY-SM, or pre-incubated with nocodazole (30 μM; b) or brefeldin A (20 μg/ml; c) for 90 min at 37 °C and then pulse-chase-labeled with BODIPY-SM as described above.

    Article Snippet: Chlorpromazine, methyl-β-cyclodextrin, genistein, nystatin, chelerythrine chloride, nocodazole, brefeldin A, SMase from Bacillus cereus , desipramine, and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) were from Sigma (Vienna).

    Techniques: Labeling, Staining, Incubation, Pulse Chase