methyl-β-cyclodextrin Millipore Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Millipore methyl β cyclodextrin mβcd
    Glycoprotein recognized by A. leucocarpus lectin is associated to lipid rafts. Murine lymph node CD4 + T cells were cultured during 48 h. Representative histograms of fluorescent cells and dot plots of proliferating cells analyzed by flow cytometry. Cells were activated via anti-CD3 mAb alone (left panels) or in the presence of anti-CD28 mAb or ALL (middle and right panels). After culture, cells were treated (blue line in histograms) or not (black line in histograms) with <t>methyl-β-cyclodextrin</t> <t>(MβCD).</t> Next, cells were incubated with biotin- ALL followed by the CyChr-streptavidin (CyChr-SA) fluorescent staining. CyChr-SA as staining control (gray line in histograms) was used. Results are representative of three independent experiments.
    Methyl β Cyclodextrin Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 774 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclodextrin mβcd/product/Millipore
    Average 95 stars, based on 774 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclodextrin mβcd - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    84
    Millipore cholesterol methyl β cyclodextrin chol mβcd
    Glycoprotein recognized by A. leucocarpus lectin is associated to lipid rafts. Murine lymph node CD4 + T cells were cultured during 48 h. Representative histograms of fluorescent cells and dot plots of proliferating cells analyzed by flow cytometry. Cells were activated via anti-CD3 mAb alone (left panels) or in the presence of anti-CD28 mAb or ALL (middle and right panels). After culture, cells were treated (blue line in histograms) or not (black line in histograms) with <t>methyl-β-cyclodextrin</t> <t>(MβCD).</t> Next, cells were incubated with biotin- ALL followed by the CyChr-streptavidin (CyChr-SA) fluorescent staining. CyChr-SA as staining control (gray line in histograms) was used. Results are representative of three independent experiments.
    Cholesterol Methyl β Cyclodextrin Chol Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cholesterol methyl β cyclodextrin chol mβcd/product/Millipore
    Average 84 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    cholesterol methyl β cyclodextrin chol mβcd - by Bioz Stars, 2020-02
    84/100 stars
      Buy from Supplier

    99
    Millipore m mβcd
    <t>MβCD</t> pretreatment diminishes amplitudes and alters the desensitization of NMDA-induced responses but does not affect kainate-induced responses
    M Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m mβcd/product/Millipore
    Average 99 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    m mβcd - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    77
    Millipore powdered mβcd
    Mean (SEM) minimum surface tensions following dynamic cycling. Lung-healthy controls achieved low (normal) surface tensions (vertical). Acute on chronic bronchiolitis (ACB) patient samples showed severe impairment in surfactant function (diagonal). ACB samples after <t>methyl-β-cyclodextrin</t> treatment <t>(MβCD</t> 40 mg/mL; horizontal) (*** p ≤ 0.001).
    Powdered Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/powdered mβcd/product/Millipore
    Average 77 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    powdered mβcd - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    99
    Millipore mβcd chol
    Mean (SEM) minimum surface tensions following dynamic cycling. Lung-healthy controls achieved low (normal) surface tensions (vertical). Acute on chronic bronchiolitis (ACB) patient samples showed severe impairment in surfactant function (diagonal). ACB samples after <t>methyl-β-cyclodextrin</t> treatment <t>(MβCD</t> 40 mg/mL; horizontal) (*** p ≤ 0.001).
    Mβcd Chol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mβcd chol/product/Millipore
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    mβcd chol - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    79
    Millipore 2 6 di o methyl β cyclodextrin dmβcd
    Mean (SEM) minimum surface tensions following dynamic cycling. Lung-healthy controls achieved low (normal) surface tensions (vertical). Acute on chronic bronchiolitis (ACB) patient samples showed severe impairment in surfactant function (diagonal). ACB samples after <t>methyl-β-cyclodextrin</t> treatment <t>(MβCD</t> 40 mg/mL; horizontal) (*** p ≤ 0.001).
    2 6 Di O Methyl β Cyclodextrin Dmβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/2 6 di o methyl β cyclodextrin dmβcd/product/Millipore
    Average 79 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    2 6 di o methyl β cyclodextrin dmβcd - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    79
    Millipore chol mβcd complex
    The effects of FC on SMC and macrophage-related gene expression. Subconfluent mouse SMCs were pretreated overnight with or without an ACAT inhibitor F-1394 (1 μM), then treated with (Chol, Chol + ACAT inhibitor) or without (Control, ACAT inhibitor alone) <t>Chol:MβCD</t> (10 μg/ml) in 0.2% BSA (72 h) in the presence of F-1394. Total RNA was extracted and subjected to QRT-PCR analysis. All data are averages ± SEM from independent duplicates. * ,α-actin was not detected. †, P
    Chol Mβcd Complex, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chol mβcd complex/product/Millipore
    Average 79 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    chol mβcd complex - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    79
    Millipore methylβ cyclodextrin mβcd
    The effects of FC on SMC and macrophage-related gene expression. Subconfluent mouse SMCs were pretreated overnight with or without an ACAT inhibitor F-1394 (1 μM), then treated with (Chol, Chol + ACAT inhibitor) or without (Control, ACAT inhibitor alone) <t>Chol:MβCD</t> (10 μg/ml) in 0.2% BSA (72 h) in the presence of F-1394. Total RNA was extracted and subjected to QRT-PCR analysis. All data are averages ± SEM from independent duplicates. * ,α-actin was not detected. †, P
    Methylβ Cyclodextrin Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methylβ cyclodextrin mβcd/product/Millipore
    Average 79 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    methylβ cyclodextrin mβcd - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    79
    Millipore lipid raft inhibitor methyl β cyclodextrin
    The lipid raft disruptor, <t>methyl-β-cyclodextrin</t> (MCD), inhibits TNF-α secretion induced by LPS but does not affect TNF-α secretion induced by cpTi particles with adherent PAMPs. RAW264.7 cells were pre-incubated with the indicated
    Lipid Raft Inhibitor Methyl β Cyclodextrin, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipid raft inhibitor methyl β cyclodextrin/product/Millipore
    Average 79 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    lipid raft inhibitor methyl β cyclodextrin - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    76
    Millipore lipid raft disruptor methyl β cyclodextrin
    Investigation of the potential N -acylethanolamine molecular targets in N1E-115 cells. N1E-115 cells express cannabinoid receptor CB 1 but not CB 2 , G-protein coupled receptor GPR55, vanilloid receptor TRPV1 and nuclear receptors PPARα and PPARγ ( A ). Detection of mRNA was performed by RT-PCR using mouse brain, spleen and liver as control. The blots are representative of three. Cytotoxicity of AEA (10 µM) ( B ), URB597 (10 µM) ( C ) and AEA + URB597 ( D ) was not significantly affected by CB 1 receptor antagonist (AM251 - 0.1 and 1 µM), TRPV1 receptor antagonist (capsazepine - 10 and 100 nM), PPAR's receptor antagonists (GW6471 and T0070907 - 0.1 and 1 µM) and GPR55 receptor antagonist (cannabidiol, CBD - 0.1 and 1 µM). N1E-115 cells were seeded 5h before treatment (2000 cells/well in microwells) and incubated with AEA alone (10 µM), URB597 alone (10 µM) and combinations of these two molecules. Antagonists were added 1h prior to the addition of AEA and/or URB597. A MTT test was used to evaluate the percentage of viable cells remaining after 72h. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Disruption of lipid rafts inhibits AEA and URB597 mediated effects on N1E-115 cell viability ( E ). Cells were preincubated with <t>methyl-β-cyclodextrin</t> (MCD, 1mM) for 1h prior to the addition of 20 µM of AEA and/or URB597. Cell viability after 72h was assessed with a MTT test. Methyl-β-cyclodextrin had no effect by itself. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Significantly different (**P
    Lipid Raft Disruptor Methyl β Cyclodextrin, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipid raft disruptor methyl β cyclodextrin/product/Millipore
    Average 76 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    lipid raft disruptor methyl β cyclodextrin - by Bioz Stars, 2020-02
    76/100 stars
      Buy from Supplier

    79
    Millipore methyl β cyclocextrin mβcd
    Investigation of the potential N -acylethanolamine molecular targets in N1E-115 cells. N1E-115 cells express cannabinoid receptor CB 1 but not CB 2 , G-protein coupled receptor GPR55, vanilloid receptor TRPV1 and nuclear receptors PPARα and PPARγ ( A ). Detection of mRNA was performed by RT-PCR using mouse brain, spleen and liver as control. The blots are representative of three. Cytotoxicity of AEA (10 µM) ( B ), URB597 (10 µM) ( C ) and AEA + URB597 ( D ) was not significantly affected by CB 1 receptor antagonist (AM251 - 0.1 and 1 µM), TRPV1 receptor antagonist (capsazepine - 10 and 100 nM), PPAR's receptor antagonists (GW6471 and T0070907 - 0.1 and 1 µM) and GPR55 receptor antagonist (cannabidiol, CBD - 0.1 and 1 µM). N1E-115 cells were seeded 5h before treatment (2000 cells/well in microwells) and incubated with AEA alone (10 µM), URB597 alone (10 µM) and combinations of these two molecules. Antagonists were added 1h prior to the addition of AEA and/or URB597. A MTT test was used to evaluate the percentage of viable cells remaining after 72h. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Disruption of lipid rafts inhibits AEA and URB597 mediated effects on N1E-115 cell viability ( E ). Cells were preincubated with <t>methyl-β-cyclodextrin</t> (MCD, 1mM) for 1h prior to the addition of 20 µM of AEA and/or URB597. Cell viability after 72h was assessed with a MTT test. Methyl-β-cyclodextrin had no effect by itself. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Significantly different (**P
    Methyl β Cyclocextrin Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclocextrin mβcd/product/Millipore
    Average 79 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclocextrin mβcd - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    77
    Millipore clathrin mediated endocytosis methyl β cyclodextrin
    Investigation of the potential N -acylethanolamine molecular targets in N1E-115 cells. N1E-115 cells express cannabinoid receptor CB 1 but not CB 2 , G-protein coupled receptor GPR55, vanilloid receptor TRPV1 and nuclear receptors PPARα and PPARγ ( A ). Detection of mRNA was performed by RT-PCR using mouse brain, spleen and liver as control. The blots are representative of three. Cytotoxicity of AEA (10 µM) ( B ), URB597 (10 µM) ( C ) and AEA + URB597 ( D ) was not significantly affected by CB 1 receptor antagonist (AM251 - 0.1 and 1 µM), TRPV1 receptor antagonist (capsazepine - 10 and 100 nM), PPAR's receptor antagonists (GW6471 and T0070907 - 0.1 and 1 µM) and GPR55 receptor antagonist (cannabidiol, CBD - 0.1 and 1 µM). N1E-115 cells were seeded 5h before treatment (2000 cells/well in microwells) and incubated with AEA alone (10 µM), URB597 alone (10 µM) and combinations of these two molecules. Antagonists were added 1h prior to the addition of AEA and/or URB597. A MTT test was used to evaluate the percentage of viable cells remaining after 72h. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Disruption of lipid rafts inhibits AEA and URB597 mediated effects on N1E-115 cell viability ( E ). Cells were preincubated with <t>methyl-β-cyclodextrin</t> (MCD, 1mM) for 1h prior to the addition of 20 µM of AEA and/or URB597. Cell viability after 72h was assessed with a MTT test. Methyl-β-cyclodextrin had no effect by itself. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Significantly different (**P
    Clathrin Mediated Endocytosis Methyl β Cyclodextrin, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/clathrin mediated endocytosis methyl β cyclodextrin/product/Millipore
    Average 77 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    clathrin mediated endocytosis methyl β cyclodextrin - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    87
    Millipore bmnpv infections mβcd
    Effect of macropinocytosis inhibitor treatment on <t>BmNPV</t> infection in Sf21 cells. Sf21 cells were mock-treated or pretreated with different inhibitors (Ehop, Rot, or Lat) for 60 min, then treated with 0.25 mM <t>MβCD</t> or PBS (CTRL) for 30 min and subsequently infected with vBmBac-ph-egfp for 2 h at a MOI of 30. ( A ) Representative images of fluorescence expression in Sf21 cells infected with BmNPV at 72 h p.i. Scale bars, 100 µm. ( B ) Flow cytometry analysis of BmNPV infection in Sf21 cells. (*** p
    Bmnpv Infections Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bmnpv infections mβcd/product/Millipore
    Average 87 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    bmnpv infections mβcd - by Bioz Stars, 2020-02
    87/100 stars
      Buy from Supplier

    82
    Millipore methyl β cyclodextran mβcd
    Effect of macropinocytosis inhibitor treatment on <t>BmNPV</t> infection in Sf21 cells. Sf21 cells were mock-treated or pretreated with different inhibitors (Ehop, Rot, or Lat) for 60 min, then treated with 0.25 mM <t>MβCD</t> or PBS (CTRL) for 30 min and subsequently infected with vBmBac-ph-egfp for 2 h at a MOI of 30. ( A ) Representative images of fluorescence expression in Sf21 cells infected with BmNPV at 72 h p.i. Scale bars, 100 µm. ( B ) Flow cytometry analysis of BmNPV infection in Sf21 cells. (*** p
    Methyl β Cyclodextran Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 82/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclodextran mβcd/product/Millipore
    Average 82 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclodextran mβcd - by Bioz Stars, 2020-02
    82/100 stars
      Buy from Supplier

    99
    Millipore mβcd cholesterol complex
    Increased cholesterol promotes association of Aβ to neurons. (A,B) Confocal microscopy images of hippocampal neurons exhibiting fluorescence for MAP-2 and demonstrating the distribution of Aβ 42 -FAM aggregates (green) on the cell membrane after treatment with <t>MβCD-Cholesterol</t> complex (200 μM) for 20 min. (A1–A3,B1–B3) ROIs of representative neurites show the overall distribution of Aβ (1 μM, after 1 h) on neuronal processes in more detail. (C,D) Representative traces of primary processes shown in (A,B) , respectively. (E,F) Quantification of Aβ clusters (number and area) in the first 20 μm of hippocampal neuron primary processes showing that both parameters were increased after treatment with MβCD-Cholesterol complex (200 μM) for 20 min. The bars represent the mean ± SEM. Asterisks denote: ∗∗ p
    Mβcd Cholesterol Complex, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mβcd cholesterol complex/product/Millipore
    Average 99 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    mβcd cholesterol complex - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    83
    Millipore prepared mβcd
    Cholesterol depletion of L. infantum chagasi metacyclic promastigotes by <t>MβCD</t> treatment. (A) Representative full GC outputs of sterols extracted from untreated control metacyclic promastigotes with a load of 2 × 10 5 cell equivalents. The
    Prepared Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/prepared mβcd/product/Millipore
    Average 83 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    prepared mβcd - by Bioz Stars, 2020-02
    83/100 stars
      Buy from Supplier

    78
    Millipore mβcd complex
    Cholesterol depletion of L. infantum chagasi metacyclic promastigotes by <t>MβCD</t> treatment. (A) Representative full GC outputs of sterols extracted from untreated control metacyclic promastigotes with a load of 2 × 10 5 cell equivalents. The
    Mβcd Complex, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mβcd complex/product/Millipore
    Average 78 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mβcd complex - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    95
    Millipore cholesterol mβcd inclusion complexes
    MHV-cell binding after cholesterol extraction or supplementation. HeLa (−) and HeLa-CEACAM (+) cells were cultured in SFM with the indicated doses of <t>MβCD</t> (CD) or cholesterol-MβCD (CHOL) complexes for 30 min at 37°C. Cells were subsequently rinsed and incubated at 4°C for 2 h with 35 S-labeled MHV strain A59 (∼5 × 10 5 cpm). Unadsorbed virions were rinsed away, and cell-associated radioactivity was calculated as a percentage of the total 35 S-labeled virus added.
    Cholesterol Mβcd Inclusion Complexes, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cholesterol mβcd inclusion complexes/product/Millipore
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    cholesterol mβcd inclusion complexes - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    95
    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: 95/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclodextrin mbcd/product/Millipore
    Average 95 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclodextrin mbcd - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    78
    Millipore human nk cells methyl β cyclodextrin
    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.
    Human Nk Cells Methyl β Cyclodextrin, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human nk cells methyl β cyclodextrin/product/Millipore
    Average 78 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    human nk cells methyl β cyclodextrin - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    79
    Millipore cholesterol depleting agents mβcd
    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.
    Cholesterol Depleting Agents Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cholesterol depleting agents mβcd/product/Millipore
    Average 79 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    cholesterol depleting agents mβcd - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    77
    Millipore cholesterol saturated mβcd
    NADPH oxidase assembly and activity in a reconstituted cell-free system depends on cholesterol. ( A ) Control or <t>mβCD-treated</t> purified macrophage membranes were extracted in TX-100 or Brij-58, and distribution of cyt b 558 subunits in the detergent-insoluble pellet (P) and soluble fraction (S) was determined by Western blotting. ( B ) Superoxide production of control or mβCD-extracted purified macrophage membranes in the reconstituted amphiphile-activated system as measured by cytochrome c reduction. Results are expressed as percent cytochrome c reduction of control, and represent mean and s.d. of four independent experiments. Superoxide production of control membranes corresponded to 15.9 mol superoxide/mol cyt b 558 heme/s. ( C ) Control or mβCD-extracted membranes mixed with cytosolic subunits were pelleted, and association of p67phox, p47phox, and Rac1 with membrane determined by lysis in Laemmli buffer and Western blotting. ( D ) Distribution of cytosolic subunits in detergent-insoluble pellet (P) and soluble fraction (S) after extraction of control membranes in Brij-58.
    Cholesterol Saturated Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cholesterol saturated mβcd/product/Millipore
    Average 77 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    cholesterol saturated mβcd - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    95
    Millipore heptakis 2 6 di o methyl β cyclodextrin
    NADPH oxidase assembly and activity in a reconstituted cell-free system depends on cholesterol. ( A ) Control or <t>mβCD-treated</t> purified macrophage membranes were extracted in TX-100 or Brij-58, and distribution of cyt b 558 subunits in the detergent-insoluble pellet (P) and soluble fraction (S) was determined by Western blotting. ( B ) Superoxide production of control or mβCD-extracted purified macrophage membranes in the reconstituted amphiphile-activated system as measured by cytochrome c reduction. Results are expressed as percent cytochrome c reduction of control, and represent mean and s.d. of four independent experiments. Superoxide production of control membranes corresponded to 15.9 mol superoxide/mol cyt b 558 heme/s. ( C ) Control or mβCD-extracted membranes mixed with cytosolic subunits were pelleted, and association of p67phox, p47phox, and Rac1 with membrane determined by lysis in Laemmli buffer and Western blotting. ( D ) Distribution of cytosolic subunits in detergent-insoluble pellet (P) and soluble fraction (S) after extraction of control membranes in Brij-58.
    Heptakis 2 6 Di O Methyl β Cyclodextrin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heptakis 2 6 di o methyl β cyclodextrin/product/Millipore
    Average 95 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    heptakis 2 6 di o methyl β cyclodextrin - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    95
    Millipore heptakis 2 3 6 tri o methyl β cyclodextrin tmβcd
    NADPH oxidase assembly and activity in a reconstituted cell-free system depends on cholesterol. ( A ) Control or <t>mβCD-treated</t> purified macrophage membranes were extracted in TX-100 or Brij-58, and distribution of cyt b 558 subunits in the detergent-insoluble pellet (P) and soluble fraction (S) was determined by Western blotting. ( B ) Superoxide production of control or mβCD-extracted purified macrophage membranes in the reconstituted amphiphile-activated system as measured by cytochrome c reduction. Results are expressed as percent cytochrome c reduction of control, and represent mean and s.d. of four independent experiments. Superoxide production of control membranes corresponded to 15.9 mol superoxide/mol cyt b 558 heme/s. ( C ) Control or mβCD-extracted membranes mixed with cytosolic subunits were pelleted, and association of p67phox, p47phox, and Rac1 with membrane determined by lysis in Laemmli buffer and Western blotting. ( D ) Distribution of cytosolic subunits in detergent-insoluble pellet (P) and soluble fraction (S) after extraction of control membranes in Brij-58.
    Heptakis 2 3 6 Tri O Methyl β Cyclodextrin Tmβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heptakis 2 3 6 tri o methyl β cyclodextrin tmβcd/product/Millipore
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    heptakis 2 3 6 tri o methyl β cyclodextrin tmβcd - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    93
    Millipore methyl β cyclo dextrin
    Surface expression of HA-CB1F238L compared with HA-CB1wt after inhibition of clathrin coated pit or caveolae mediated endocytosis. (A) HEK293 cells stably expressing HA-CB1wt or HA-CB1F238L were treated either for 15 min with 20 μM PitStop2™ to inhibit clathrin coated pit endocytosis or for 30 min with 5 mM <t>methyl-β-cyclo-dextrin</t> (MβCD) to inhibit caveolae mediated endocytosis. Surface expression was analyzed by a trypsin protection assay. (B) Only MβCD treatment significantly increased surface expression of CB1F238L receptor. (Two way ANOVA and Bonferroni’s post hoc test. Data are presented as the mean ± SEM of n = 4 independent experiments. Surface HA-CB1 was calculated and expressed as percent of total HA-CB1 as described in “Materials and Methods” section * p
    Methyl β Cyclo Dextrin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclo dextrin/product/Millipore
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclo dextrin - by Bioz Stars, 2020-02
    93/100 stars
      Buy from Supplier

    76
    Millipore methyl β cyclodextrine mecd
    Surface expression of HA-CB1F238L compared with HA-CB1wt after inhibition of clathrin coated pit or caveolae mediated endocytosis. (A) HEK293 cells stably expressing HA-CB1wt or HA-CB1F238L were treated either for 15 min with 20 μM PitStop2™ to inhibit clathrin coated pit endocytosis or for 30 min with 5 mM <t>methyl-β-cyclo-dextrin</t> (MβCD) to inhibit caveolae mediated endocytosis. Surface expression was analyzed by a trypsin protection assay. (B) Only MβCD treatment significantly increased surface expression of CB1F238L receptor. (Two way ANOVA and Bonferroni’s post hoc test. Data are presented as the mean ± SEM of n = 4 independent experiments. Surface HA-CB1 was calculated and expressed as percent of total HA-CB1 as described in “Materials and Methods” section * p
    Methyl β Cyclodextrine Mecd, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclodextrine mecd/product/Millipore
    Average 76 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclodextrine mecd - by Bioz Stars, 2020-02
    76/100 stars
      Buy from Supplier

    79
    Millipore methyl β cyclodextrine mcd
    Surface expression of HA-CB1F238L compared with HA-CB1wt after inhibition of clathrin coated pit or caveolae mediated endocytosis. (A) HEK293 cells stably expressing HA-CB1wt or HA-CB1F238L were treated either for 15 min with 20 μM PitStop2™ to inhibit clathrin coated pit endocytosis or for 30 min with 5 mM <t>methyl-β-cyclo-dextrin</t> (MβCD) to inhibit caveolae mediated endocytosis. Surface expression was analyzed by a trypsin protection assay. (B) Only MβCD treatment significantly increased surface expression of CB1F238L receptor. (Two way ANOVA and Bonferroni’s post hoc test. Data are presented as the mean ± SEM of n = 4 independent experiments. Surface HA-CB1 was calculated and expressed as percent of total HA-CB1 as described in “Materials and Methods” section * p
    Methyl β Cyclodextrine Mcd, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclodextrine mcd/product/Millipore
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclodextrine mcd - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    79
    Millipore beta methyl ciclodextrine
    Surface expression of HA-CB1F238L compared with HA-CB1wt after inhibition of clathrin coated pit or caveolae mediated endocytosis. (A) HEK293 cells stably expressing HA-CB1wt or HA-CB1F238L were treated either for 15 min with 20 μM PitStop2™ to inhibit clathrin coated pit endocytosis or for 30 min with 5 mM <t>methyl-β-cyclo-dextrin</t> (MβCD) to inhibit caveolae mediated endocytosis. Surface expression was analyzed by a trypsin protection assay. (B) Only MβCD treatment significantly increased surface expression of CB1F238L receptor. (Two way ANOVA and Bonferroni’s post hoc test. Data are presented as the mean ± SEM of n = 4 independent experiments. Surface HA-CB1 was calculated and expressed as percent of total HA-CB1 as described in “Materials and Methods” section * p
    Beta Methyl Ciclodextrine, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/beta methyl ciclodextrine/product/Millipore
    Average 79 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    beta methyl ciclodextrine - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    78
    Millipore methyl β cyclodetritrin
    Surface expression of HA-CB1F238L compared with HA-CB1wt after inhibition of clathrin coated pit or caveolae mediated endocytosis. (A) HEK293 cells stably expressing HA-CB1wt or HA-CB1F238L were treated either for 15 min with 20 μM PitStop2™ to inhibit clathrin coated pit endocytosis or for 30 min with 5 mM <t>methyl-β-cyclo-dextrin</t> (MβCD) to inhibit caveolae mediated endocytosis. Surface expression was analyzed by a trypsin protection assay. (B) Only MβCD treatment significantly increased surface expression of CB1F238L receptor. (Two way ANOVA and Bonferroni’s post hoc test. Data are presented as the mean ± SEM of n = 4 independent experiments. Surface HA-CB1 was calculated and expressed as percent of total HA-CB1 as described in “Materials and Methods” section * p
    Methyl β Cyclodetritrin, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclodetritrin/product/Millipore
    Average 78 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclodetritrin - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    99
    Millipore methyl β cyclodextran
    Surface expression of HA-CB1F238L compared with HA-CB1wt after inhibition of clathrin coated pit or caveolae mediated endocytosis. (A) HEK293 cells stably expressing HA-CB1wt or HA-CB1F238L were treated either for 15 min with 20 μM PitStop2™ to inhibit clathrin coated pit endocytosis or for 30 min with 5 mM <t>methyl-β-cyclo-dextrin</t> (MβCD) to inhibit caveolae mediated endocytosis. Surface expression was analyzed by a trypsin protection assay. (B) Only MβCD treatment significantly increased surface expression of CB1F238L receptor. (Two way ANOVA and Bonferroni’s post hoc test. Data are presented as the mean ± SEM of n = 4 independent experiments. Surface HA-CB1 was calculated and expressed as percent of total HA-CB1 as described in “Materials and Methods” section * p
    Methyl β Cyclodextran, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclodextran/product/Millipore
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclodextran - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    75
    Millipore caveoli
    Surface expression of HA-CB1F238L compared with HA-CB1wt after inhibition of clathrin coated pit or caveolae mediated endocytosis. (A) HEK293 cells stably expressing HA-CB1wt or HA-CB1F238L were treated either for 15 min with 20 μM PitStop2™ to inhibit clathrin coated pit endocytosis or for 30 min with 5 mM <t>methyl-β-cyclo-dextrin</t> (MβCD) to inhibit caveolae mediated endocytosis. Surface expression was analyzed by a trypsin protection assay. (B) Only MβCD treatment significantly increased surface expression of CB1F238L receptor. (Two way ANOVA and Bonferroni’s post hoc test. Data are presented as the mean ± SEM of n = 4 independent experiments. Surface HA-CB1 was calculated and expressed as percent of total HA-CB1 as described in “Materials and Methods” section * p
    Caveoli, supplied by Millipore, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caveoli/product/Millipore
    Average 75 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    caveoli - by Bioz Stars, 2020-02
    75/100 stars
      Buy from Supplier

    Image Search Results


    Glycoprotein recognized by A. leucocarpus lectin is associated to lipid rafts. Murine lymph node CD4 + T cells were cultured during 48 h. Representative histograms of fluorescent cells and dot plots of proliferating cells analyzed by flow cytometry. Cells were activated via anti-CD3 mAb alone (left panels) or in the presence of anti-CD28 mAb or ALL (middle and right panels). After culture, cells were treated (blue line in histograms) or not (black line in histograms) with methyl-β-cyclodextrin (MβCD). Next, cells were incubated with biotin- ALL followed by the CyChr-streptavidin (CyChr-SA) fluorescent staining. CyChr-SA as staining control (gray line in histograms) was used. Results are representative of three independent experiments.

    Journal: Immunity, Inflammation and Disease

    Article Title: Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4+ T cells

    doi: 10.1002/iid3.58

    Figure Lengend Snippet: Glycoprotein recognized by A. leucocarpus lectin is associated to lipid rafts. Murine lymph node CD4 + T cells were cultured during 48 h. Representative histograms of fluorescent cells and dot plots of proliferating cells analyzed by flow cytometry. Cells were activated via anti-CD3 mAb alone (left panels) or in the presence of anti-CD28 mAb or ALL (middle and right panels). After culture, cells were treated (blue line in histograms) or not (black line in histograms) with methyl-β-cyclodextrin (MβCD). Next, cells were incubated with biotin- ALL followed by the CyChr-streptavidin (CyChr-SA) fluorescent staining. CyChr-SA as staining control (gray line in histograms) was used. Results are representative of three independent experiments.

    Article Snippet: Bovine serum albumin fraction V (BSA) ≥95% purity, RPMI-1640 culture medium, Coomassie brilliant blue R-250, trypan blue, Triton X-100 Ultra-pure, polyoxyethylenesorbitan monolaurate (Tween-20), dimethyl sulfoxide, methyl-β-cyclodextrin (MβCD), peroxidase-labeled extravidin, saponin, biotin-labeled cholera toxin B subunit, brefeldin-A from Penicillum brefeldianum , OptiPrep, ethylenediamine tetraacetic acid (EDTA), sodium azide, Trizma base, HEPES, and other salts were from Sigma–Aldrich (St. Louis, MO).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Incubation, Staining

    PrP ∗ Traffics to the Plasma Membrane during RESET (A) Time-lapse images of YFP-PrP ∗ -expressing cells treated with TG + methyl-β-cyclodextrin (MβCD) (top) or TG alone (bottom) taken at two different focal planes. Mid-cell indicates a focal plane at the widest point of the nucleus and coverslip indicates a focal plane close to the coverslip where the largest portion of the plasma membrane is in focus. (B) YFP-PrP ∗ cells were untreated or treated with TG and MβCD and then fixed after 90 min and stained with anti-GFP antibody without permeabilization to detect cell surface YFP-PrP ∗ . (C) Antibody uptake assay for internalization of YFP-PrP ∗ from the plasma membrane. YFP-PrP ∗ -expressing or untransfected (Untf’d) cells were treated for 1 hr with TG in the presence of leupeptin and either anti-GFP or anti-Myc. The cells were then washed, fixed, permeabilized, and stained with Cy5-conjugated secondary antibody to detect internalized antibody. Scale bars, 10 μm.

    Journal: Cell

    Article Title: ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway

    doi: 10.1016/j.cell.2014.06.026

    Figure Lengend Snippet: PrP ∗ Traffics to the Plasma Membrane during RESET (A) Time-lapse images of YFP-PrP ∗ -expressing cells treated with TG + methyl-β-cyclodextrin (MβCD) (top) or TG alone (bottom) taken at two different focal planes. Mid-cell indicates a focal plane at the widest point of the nucleus and coverslip indicates a focal plane close to the coverslip where the largest portion of the plasma membrane is in focus. (B) YFP-PrP ∗ cells were untreated or treated with TG and MβCD and then fixed after 90 min and stained with anti-GFP antibody without permeabilization to detect cell surface YFP-PrP ∗ . (C) Antibody uptake assay for internalization of YFP-PrP ∗ from the plasma membrane. YFP-PrP ∗ -expressing or untransfected (Untf’d) cells were treated for 1 hr with TG in the presence of leupeptin and either anti-GFP or anti-Myc. The cells were then washed, fixed, permeabilized, and stained with Cy5-conjugated secondary antibody to detect internalized antibody. Scale bars, 10 μm.

    Article Snippet: Drug Treatments The following reagents were used at working concentrations as listed, unless specified: 0.1 μM thapsigargin (EMD 586005), 0.5 mM 1,3-dithiothreitol (Roche 10708984001), 125 μM leupeptin (Sigma L5793), 250 nM bafilomycin A1 (LC Laboratories B-1080), 5 mM methyl-β-cyclodextrin (Sigma C4555), 1 μM brefeldin A (Sigma B7651).

    Techniques: Expressing, Staining

    Rafts depletion induces endogenous EGFR-PTPH1 interaction, EGFR dephopshorylation, and its intracellular arrest in MDA-MB-468 TNBC cells. a Immunofluorescence assay (IF) was performed by using anti-EGFR (green) and anti-GM1 (red) antibodies to reveal the endogenous EGFR-rafts colocalization, shown in yellow (merge). Nuclei were DAPI labeled (blue). b Raft (R) and non-raft (NR) fractions derived from Methyl-β-cyclodextrin (MβCD)-treated and untreated cells were used for immunoblot assay with anti-pEGFR (Y1173) (indicated as pEGFR) and anti-EGFR antibodies, to test activated and total EGFR expression in rafts compartment, respectively. Anti-transferrin and anti-GM1 antibodies were used as a fraction markers. c Cells have been activated with EGF ligand for the times indicated, in the presence or absence of MβCD: the expression of phospho-EGFR at tyrosine 1173 and 1068 residues and total EGFR was determined in whole cell extracts by immunoblot analysis using the specific indicated antibodies. d – f MDA-MB-468 cells were treated with MβCD and stimulated with EGF for 60 min: control or anti-PTPH1 antibody immunoprecipitates were probed with anti-EGFR, to detect the EGFR-PTPH1 binding, and with the anti-PTPH1 antibody, to show PTPH1 immunoprecipitated protein levels. The inputs indicated in the panel shows 5% of each total lysate d . Relative EGFR extracellular expression (EGFR EC ) was evaluated by FACS e . IF assay was performed by using anti-EGFR (red) antibody to reveal the endogenous EGFR intracellular localization. Nuclei were DAPI labeled (blue). White arrows indicated peri-nuclear EGFR localization in EGF stimulated MβCD-treated cells ( f ). a , f Representative single plane confocal IF images captured using a × 60 oil objective. Scale bar: 10 μm. In both b and c , western blotting against the anti-β-actin was used as a loading control. All data are representative of at least three independent experiments, each in triplicate. Results shown in e are expressed as the means average deviations and P -values were calculated using Student’s T -test (i.e., ns, not significant P > 0.05, ** P ≤ 0.01)

    Journal: Oncogenesis

    Article Title: NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest

    doi: 10.1038/s41389-018-0051-9

    Figure Lengend Snippet: Rafts depletion induces endogenous EGFR-PTPH1 interaction, EGFR dephopshorylation, and its intracellular arrest in MDA-MB-468 TNBC cells. a Immunofluorescence assay (IF) was performed by using anti-EGFR (green) and anti-GM1 (red) antibodies to reveal the endogenous EGFR-rafts colocalization, shown in yellow (merge). Nuclei were DAPI labeled (blue). b Raft (R) and non-raft (NR) fractions derived from Methyl-β-cyclodextrin (MβCD)-treated and untreated cells were used for immunoblot assay with anti-pEGFR (Y1173) (indicated as pEGFR) and anti-EGFR antibodies, to test activated and total EGFR expression in rafts compartment, respectively. Anti-transferrin and anti-GM1 antibodies were used as a fraction markers. c Cells have been activated with EGF ligand for the times indicated, in the presence or absence of MβCD: the expression of phospho-EGFR at tyrosine 1173 and 1068 residues and total EGFR was determined in whole cell extracts by immunoblot analysis using the specific indicated antibodies. d – f MDA-MB-468 cells were treated with MβCD and stimulated with EGF for 60 min: control or anti-PTPH1 antibody immunoprecipitates were probed with anti-EGFR, to detect the EGFR-PTPH1 binding, and with the anti-PTPH1 antibody, to show PTPH1 immunoprecipitated protein levels. The inputs indicated in the panel shows 5% of each total lysate d . Relative EGFR extracellular expression (EGFR EC ) was evaluated by FACS e . IF assay was performed by using anti-EGFR (red) antibody to reveal the endogenous EGFR intracellular localization. Nuclei were DAPI labeled (blue). White arrows indicated peri-nuclear EGFR localization in EGF stimulated MβCD-treated cells ( f ). a , f Representative single plane confocal IF images captured using a × 60 oil objective. Scale bar: 10 μm. In both b and c , western blotting against the anti-β-actin was used as a loading control. All data are representative of at least three independent experiments, each in triplicate. Results shown in e are expressed as the means average deviations and P -values were calculated using Student’s T -test (i.e., ns, not significant P > 0.05, ** P ≤ 0.01)

    Article Snippet: Cells were treated with the following compounds: 5 mM MβCD (Sigma-Aldrich, Catalog number C4555); 3 μM GEF (Iressa, Selleckem, Houston, TX, USA; Catalog number ZD1839), 100 nM EGF Ligand (EGF; Gibco, Life Technologies, Carlsbad, CA, USA; Catalog number PHG0315); 10 μM of GSI IX (DAPT) (Calbiochem, Darmstadt, Germany; Catalog number 565770).

    Techniques: Multiple Displacement Amplification, Immunofluorescence, Labeling, Derivative Assay, Expressing, Binding Assay, Immunoprecipitation, FACS, Western Blot

    Accurate NSOM imaging and quantification of GM1/CD59 on Jurkat T cells pre-fixed by FA of various concentrations and at various temperatures. (A) NSOM images (merged from the NSOM fluorescence images in red and the corresponding topographic images in gray) show the size enlargement of GM1 clusters from nanoclusters to microclusters with the decrease in fixation strength from 2% FA to 0% no fixation. (B, C) The NSOM images of GM1 from representatives of Jurkat cells pre-fixed by 2% FA at 37°C (B) or treated with 10 mM MβCD for 30 min followed by pre-fixation with 2% FA at 4°C and fluorescence staining (C). (D) Dot graph (bottom) and X-Y plot (top; SEM±SD) of the diameters (FWHM) of the GM1 micro/nanoclusters on cells (5 cells/group) pre-fixed with 0%, 0.1%, 0.5%, 2%, 10% FA at 4°C, and 2% FA at 37°C, respectively. (E, F) NSOM images of CD59 on a representative cell (Left) pre-fixed by 10% FA at 4°C (E) or at 37°C (F) and the boxed part (right) of it; (G) The fluorescence profiles of the cross sections across the centers of the two CD59 nanoclusters indicated by numbers 1 and 2 in Fig. E (left of Fig. G) or F (right). (H) Dot (left) and bar (right; Mean±SD) graphs of the diameters (FWHM) of the CD59 nanoclusters on cells (5 cells/group) pre-fixed with 10% FA at 4°C and at 37°C, respectively). Scan size: (A: left to right) 14×14 μm 2 ; 15.5×15.5 μm 2 ; 17×17 μm 2 . (B, C) 20×20 μm 2 ; (E: left to right) 18×18 μm 2 ; ∼4×4 μm 2 ; (F: left to right) 15×15 μm 2 ; ∼4×4 μm 2 . Scale bar/resolution: (A–C, and left panels of E and F) 1 μm/500×500 pixel 2 ; (right panels of E and F) 500 nm/300×300 pixel 2 . Integration time: (all) 30 ms.

    Journal: PLoS ONE

    Article Title: Cold Induces Micro- and Nano-Scale Reorganization of Lipid Raft Markers at Mounds of T-Cell Membrane Fluctuations

    doi: 10.1371/journal.pone.0005386

    Figure Lengend Snippet: Accurate NSOM imaging and quantification of GM1/CD59 on Jurkat T cells pre-fixed by FA of various concentrations and at various temperatures. (A) NSOM images (merged from the NSOM fluorescence images in red and the corresponding topographic images in gray) show the size enlargement of GM1 clusters from nanoclusters to microclusters with the decrease in fixation strength from 2% FA to 0% no fixation. (B, C) The NSOM images of GM1 from representatives of Jurkat cells pre-fixed by 2% FA at 37°C (B) or treated with 10 mM MβCD for 30 min followed by pre-fixation with 2% FA at 4°C and fluorescence staining (C). (D) Dot graph (bottom) and X-Y plot (top; SEM±SD) of the diameters (FWHM) of the GM1 micro/nanoclusters on cells (5 cells/group) pre-fixed with 0%, 0.1%, 0.5%, 2%, 10% FA at 4°C, and 2% FA at 37°C, respectively. (E, F) NSOM images of CD59 on a representative cell (Left) pre-fixed by 10% FA at 4°C (E) or at 37°C (F) and the boxed part (right) of it; (G) The fluorescence profiles of the cross sections across the centers of the two CD59 nanoclusters indicated by numbers 1 and 2 in Fig. E (left of Fig. G) or F (right). (H) Dot (left) and bar (right; Mean±SD) graphs of the diameters (FWHM) of the CD59 nanoclusters on cells (5 cells/group) pre-fixed with 10% FA at 4°C and at 37°C, respectively). Scan size: (A: left to right) 14×14 μm 2 ; 15.5×15.5 μm 2 ; 17×17 μm 2 . (B, C) 20×20 μm 2 ; (E: left to right) 18×18 μm 2 ; ∼4×4 μm 2 ; (F: left to right) 15×15 μm 2 ; ∼4×4 μm 2 . Scale bar/resolution: (A–C, and left panels of E and F) 1 μm/500×500 pixel 2 ; (right panels of E and F) 500 nm/300×300 pixel 2 . Integration time: (all) 30 ms.

    Article Snippet: The reagents/antibodies used are as follows: biotinylated CTB, FITC-conjuaged CTB, PHA, and MβCD were purchased from Sigma-Aldrich (St Louis, MO, USA); fluorescent QD655-conjugated streptavidin, QD605-conjugated strepavidin, and QD655-conjugated goat anti-mouse IgG antibody (H+L) were from Invitrogen (Carlsbad, CA, USA); biotinylated anti-CD59 antibody and FITC-conjugated CD59 were from EXBIO Praha (Vestec, Czech Republic) or BD Biosciences (San Jose, CA, USA); FITC-conjugated CD71 antibody from BD Biosciences.

    Techniques: Imaging, Fluorescence, Staining, Mass Spectrometry

    Imatinib or masitinib induces c-Kit internalization and its degradation by lysosome pathway. A. For analysis of internalization, UT-7/Epo cells were pre-incubated with 50 µM of methyl-β-cyclodextrine (MβCD) or vehicle (V) for 30 min then treated during 4 hours with imatinib (grey bars) or masitinib (black bars) or Epo alone (white bars). B. For the analysis of proteasomal degradation, UT-7/Epo cells were incubated with 1 UI/ml of Epo or 50 ng/ml of SCF (hatched bars), and 50 µM of LLnL (L) or vehicle (V) for 20 min at 37°C before treatment with 2 µM IM (grey bars) or MA (black bars) or Epo alone (white bars) for 4 h. C. For the analysis of lysosomal degradation, UT-7/Epo cells were incubated with 1 UI/ml Epo or 50 ng/ml SCF, and 100 µM methylamine (M) or vehicle (V) for 20 min before treatment with 2 µM IM (grey bars) or MA (black bars) or Epo alone (white bars) for 4 h. Expression of c-Kit by flow cytometry (cell surface) and by immunoblot. Actin was used as loading control. Data are representative of three independent experiments and the quantification of one experiment is shown.

    Journal: PLoS ONE

    Article Title: Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket

    doi: 10.1371/journal.pone.0060961

    Figure Lengend Snippet: Imatinib or masitinib induces c-Kit internalization and its degradation by lysosome pathway. A. For analysis of internalization, UT-7/Epo cells were pre-incubated with 50 µM of methyl-β-cyclodextrine (MβCD) or vehicle (V) for 30 min then treated during 4 hours with imatinib (grey bars) or masitinib (black bars) or Epo alone (white bars). B. For the analysis of proteasomal degradation, UT-7/Epo cells were incubated with 1 UI/ml of Epo or 50 ng/ml of SCF (hatched bars), and 50 µM of LLnL (L) or vehicle (V) for 20 min at 37°C before treatment with 2 µM IM (grey bars) or MA (black bars) or Epo alone (white bars) for 4 h. C. For the analysis of lysosomal degradation, UT-7/Epo cells were incubated with 1 UI/ml Epo or 50 ng/ml SCF, and 100 µM methylamine (M) or vehicle (V) for 20 min before treatment with 2 µM IM (grey bars) or MA (black bars) or Epo alone (white bars) for 4 h. Expression of c-Kit by flow cytometry (cell surface) and by immunoblot. Actin was used as loading control. Data are representative of three independent experiments and the quantification of one experiment is shown.

    Article Snippet: Methyl-β-cyclodextrine (MβCD), methylamine and cycloheximide (CHX) were purchased from Sigma Aldrich.

    Techniques: Incubation, Expressing, Flow Cytometry, Cytometry

    MβCD pretreatment diminishes amplitudes and alters the desensitization of NMDA-induced responses but does not affect kainate-induced responses

    Journal: The Journal of Physiology

    Article Title: Cholesterol modulates open probability and desensitization of NMDA receptors

    doi: 10.1113/jphysiol.2014.288209

    Figure Lengend Snippet: MβCD pretreatment diminishes amplitudes and alters the desensitization of NMDA-induced responses but does not affect kainate-induced responses

    Article Snippet: For acute cholesterol depletion by MβCD, DIV 6–7 CGCs were exposed to low-potassium medium supplemented with 5 m m MβCD (mean molecular weight 1310 g mol−1 ; Aldrich, St Louis, MO, USA) and incubated at 37°C in 5% CO2 for 1–60 min as indicated.

    Techniques:

    Cholesterol repletion restores the amplitude and desensitization of NMDAR responses in MβCD-pretreated CGCs to control values

    Journal: The Journal of Physiology

    Article Title: Cholesterol modulates open probability and desensitization of NMDA receptors

    doi: 10.1113/jphysiol.2014.288209

    Figure Lengend Snippet: Cholesterol repletion restores the amplitude and desensitization of NMDAR responses in MβCD-pretreated CGCs to control values

    Article Snippet: For acute cholesterol depletion by MβCD, DIV 6–7 CGCs were exposed to low-potassium medium supplemented with 5 m m MβCD (mean molecular weight 1310 g mol−1 ; Aldrich, St Louis, MO, USA) and incubated at 37°C in 5% CO2 for 1–60 min as indicated.

    Techniques:

    Chronic inhibition of cholesterol biosynthesis by simvastatin induces qualitatively the same effects on NMDA and AMPA/kainate receptors as acute MβCD pretreatment

    Journal: The Journal of Physiology

    Article Title: Cholesterol modulates open probability and desensitization of NMDA receptors

    doi: 10.1113/jphysiol.2014.288209

    Figure Lengend Snippet: Chronic inhibition of cholesterol biosynthesis by simvastatin induces qualitatively the same effects on NMDA and AMPA/kainate receptors as acute MβCD pretreatment

    Article Snippet: For acute cholesterol depletion by MβCD, DIV 6–7 CGCs were exposed to low-potassium medium supplemented with 5 m m MβCD (mean molecular weight 1310 g mol−1 ; Aldrich, St Louis, MO, USA) and incubated at 37°C in 5% CO2 for 1–60 min as indicated.

    Techniques: Inhibition

    Cortical 45-kDa Gαs is contained in low-density membrane and is reduced after treatment of oocytes with progesterone or MeβCD

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: Cortical 45-kDa Gαs is contained in low-density membrane and is reduced after treatment of oocytes with progesterone or MeβCD

    Article Snippet: Groups of oocytes were incubated in the indicated volumes of oocyte Ringers-HCO3 (73 m M NaCl, 10 m M NaHCO3 , 1 m M MgCl2 , 0.5 m M CaCl2 , 1 m M KCl, 25 m M Hepes, pH 7.9) in the absence or presence of indicated concentrations of progesterone (Calbiochem; diluted into oocyte Ringers-HCO3 from a 0.5 mg/ml stock solution in ethanol), 50 m M MeβCD (Sigma), or 50 IU human chorionic gonadotropin (hCG) (Sigma).

    Techniques:

    Progesterone- and MeβCD-stimulated increases in oocyte 39-kDa Mos are inhibited by cycloheximide

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: Progesterone- and MeβCD-stimulated increases in oocyte 39-kDa Mos are inhibited by cycloheximide

    Article Snippet: Groups of oocytes were incubated in the indicated volumes of oocyte Ringers-HCO3 (73 m M NaCl, 10 m M NaHCO3 , 1 m M MgCl2 , 0.5 m M CaCl2 , 1 m M KCl, 25 m M Hepes, pH 7.9) in the absence or presence of indicated concentrations of progesterone (Calbiochem; diluted into oocyte Ringers-HCO3 from a 0.5 mg/ml stock solution in ethanol), 50 m M MeβCD (Sigma), or 50 IU human chorionic gonadotropin (hCG) (Sigma).

    Techniques:

    The steroid synthesis inhibitor, aminoglutethimide, does not block oocyte maturation stimulated MeβCD

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: The steroid synthesis inhibitor, aminoglutethimide, does not block oocyte maturation stimulated MeβCD

    Article Snippet: Groups of oocytes were incubated in the indicated volumes of oocyte Ringers-HCO3 (73 m M NaCl, 10 m M NaHCO3 , 1 m M MgCl2 , 0.5 m M CaCl2 , 1 m M KCl, 25 m M Hepes, pH 7.9) in the absence or presence of indicated concentrations of progesterone (Calbiochem; diluted into oocyte Ringers-HCO3 from a 0.5 mg/ml stock solution in ethanol), 50 m M MeβCD (Sigma), or 50 IU human chorionic gonadotropin (hCG) (Sigma).

    Techniques: Blocking Assay

    Follicle cells are not required for phosphorylation of oocyte MAPK or GVBD induced by MeβCD

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: Follicle cells are not required for phosphorylation of oocyte MAPK or GVBD induced by MeβCD

    Article Snippet: Groups of oocytes were incubated in the indicated volumes of oocyte Ringers-HCO3 (73 m M NaCl, 10 m M NaHCO3 , 1 m M MgCl2 , 0.5 m M CaCl2 , 1 m M KCl, 25 m M Hepes, pH 7.9) in the absence or presence of indicated concentrations of progesterone (Calbiochem; diluted into oocyte Ringers-HCO3 from a 0.5 mg/ml stock solution in ethanol), 50 m M MeβCD (Sigma), or 50 IU human chorionic gonadotropin (hCG) (Sigma).

    Techniques:

    Cycloheximide blocks MAPK phosphorylation and GVBD in response to MeβCD

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: Cycloheximide blocks MAPK phosphorylation and GVBD in response to MeβCD

    Article Snippet: Groups of oocytes were incubated in the indicated volumes of oocyte Ringers-HCO3 (73 m M NaCl, 10 m M NaHCO3 , 1 m M MgCl2 , 0.5 m M CaCl2 , 1 m M KCl, 25 m M Hepes, pH 7.9) in the absence or presence of indicated concentrations of progesterone (Calbiochem; diluted into oocyte Ringers-HCO3 from a 0.5 mg/ml stock solution in ethanol), 50 m M MeβCD (Sigma), or 50 IU human chorionic gonadotropin (hCG) (Sigma).

    Techniques:

    Mean (SEM) minimum surface tensions following dynamic cycling. Lung-healthy controls achieved low (normal) surface tensions (vertical). Acute on chronic bronchiolitis (ACB) patient samples showed severe impairment in surfactant function (diagonal). ACB samples after methyl-β-cyclodextrin treatment (MβCD 40 mg/mL; horizontal) (*** p ≤ 0.001).

    Journal: Military Medicine

    Article Title: Surfactant Dysfunction in ARDS and Bronchiolitis is Repaired with Cyclodextrins

    doi: 10.1093/milmed/usx204

    Figure Lengend Snippet: Mean (SEM) minimum surface tensions following dynamic cycling. Lung-healthy controls achieved low (normal) surface tensions (vertical). Acute on chronic bronchiolitis (ACB) patient samples showed severe impairment in surfactant function (diagonal). ACB samples after methyl-β-cyclodextrin treatment (MβCD 40 mg/mL; horizontal) (*** p ≤ 0.001).

    Article Snippet: To evaluate the effect of MβCD, powdered MβCD (Sigma-Aldrich, Catalogue-Nr.

    Techniques:

    Minimum surface tension during dynamic cycle 20 with BLES containing 27 mg/mL BLES in control CBS buffer (diagonals bar), BLES with 20% w/w free fatty acid (linoleic acid) (vertical bar), and BLES with 20% w/w free fatty acid + 40 mg/mL MβCD (horizontal bar). (** p ≤ 0.01, *** p ≤ 0.001). BLES with FFA or LPC shows marked impairment, which is repaired to normal functionality in the presence of MβCD.

    Journal: Military Medicine

    Article Title: Surfactant Dysfunction in ARDS and Bronchiolitis is Repaired with Cyclodextrins

    doi: 10.1093/milmed/usx204

    Figure Lengend Snippet: Minimum surface tension during dynamic cycle 20 with BLES containing 27 mg/mL BLES in control CBS buffer (diagonals bar), BLES with 20% w/w free fatty acid (linoleic acid) (vertical bar), and BLES with 20% w/w free fatty acid + 40 mg/mL MβCD (horizontal bar). (** p ≤ 0.01, *** p ≤ 0.001). BLES with FFA or LPC shows marked impairment, which is repaired to normal functionality in the presence of MβCD.

    Article Snippet: To evaluate the effect of MβCD, powdered MβCD (Sigma-Aldrich, Catalogue-Nr.

    Techniques:

    (A) Minimum surface tensions during dynamic compression–expansion cycles of mouse lipid extract surfactant (MLES), from saline-exposed, acid-exposed, or CBS buffer containing 30 mM MβCD (horizontal) animals, * p

    Journal: Military Medicine

    Article Title: Surfactant Dysfunction in ARDS and Bronchiolitis is Repaired with Cyclodextrins

    doi: 10.1093/milmed/usx204

    Figure Lengend Snippet: (A) Minimum surface tensions during dynamic compression–expansion cycles of mouse lipid extract surfactant (MLES), from saline-exposed, acid-exposed, or CBS buffer containing 30 mM MβCD (horizontal) animals, * p

    Article Snippet: To evaluate the effect of MβCD, powdered MβCD (Sigma-Aldrich, Catalogue-Nr.

    Techniques:

    Minimum surface tensions during dynamic compression–expansion cycles of oxidized bovine lipid extract surfactant (oxBLES, cholesterol free) (white), oxBLES + 10% w/w cholesterol in control CBS buffer (checker), or buffer containing 30 mM MβCD (black).

    Journal: Military Medicine

    Article Title: Surfactant Dysfunction in ARDS and Bronchiolitis is Repaired with Cyclodextrins

    doi: 10.1093/milmed/usx204

    Figure Lengend Snippet: Minimum surface tensions during dynamic compression–expansion cycles of oxidized bovine lipid extract surfactant (oxBLES, cholesterol free) (white), oxBLES + 10% w/w cholesterol in control CBS buffer (checker), or buffer containing 30 mM MβCD (black).

    Article Snippet: To evaluate the effect of MβCD, powdered MβCD (Sigma-Aldrich, Catalogue-Nr.

    Techniques:

    The effects of FC on SMC and macrophage-related gene expression. Subconfluent mouse SMCs were pretreated overnight with or without an ACAT inhibitor F-1394 (1 μM), then treated with (Chol, Chol + ACAT inhibitor) or without (Control, ACAT inhibitor alone) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h) in the presence of F-1394. Total RNA was extracted and subjected to QRT-PCR analysis. All data are averages ± SEM from independent duplicates. * ,α-actin was not detected. †, P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading

    doi: 10.1073/pnas.1735526100

    Figure Lengend Snippet: The effects of FC on SMC and macrophage-related gene expression. Subconfluent mouse SMCs were pretreated overnight with or without an ACAT inhibitor F-1394 (1 μM), then treated with (Chol, Chol + ACAT inhibitor) or without (Control, ACAT inhibitor alone) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h) in the presence of F-1394. Total RNA was extracted and subjected to QRT-PCR analysis. All data are averages ± SEM from independent duplicates. * ,α-actin was not detected. †, P

    Article Snippet: Cholesterol was delivered to cells by using Chol:MβCD complex purchased from Sigma as “water-soluble cholesterol” (catalog no. C4951) containing ≈50 mg of cholesterol/g solid (molar ratio, 1:6 cholesterol/MβCD).

    Techniques: Expressing, Quantitative RT-PCR

    Immunostaining of SMCs for SMC and macrophage-related proteins. Subconfluent mouse SMCs were treated with (Chol) or without (Control) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h). Cells were stained with antibodies for α-actin (red stain, ×200), α-tropomyosin (brown stain, ×200), CD68 (brown stain, ×200), and Mac-2 (brown stain, ×200).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading

    doi: 10.1073/pnas.1735526100

    Figure Lengend Snippet: Immunostaining of SMCs for SMC and macrophage-related proteins. Subconfluent mouse SMCs were treated with (Chol) or without (Control) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h). Cells were stained with antibodies for α-actin (red stain, ×200), α-tropomyosin (brown stain, ×200), CD68 (brown stain, ×200), and Mac-2 (brown stain, ×200).

    Article Snippet: Cholesterol was delivered to cells by using Chol:MβCD complex purchased from Sigma as “water-soluble cholesterol” (catalog no. C4951) containing ≈50 mg of cholesterol/g solid (molar ratio, 1:6 cholesterol/MβCD).

    Techniques: Immunostaining, Staining

    QRT-PCR determination of SMC and macrophage-related gene expression in SMCs. Subconfluent mouse SMCs were treated with (Chol) or without (Control) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h). In C , control or cholesterol-loaded cells were treated with 30 ng/ml TNF-α for 2 h after cholesterol loading. Total RNA was extracted and subjected to QRT-PCR analysis of smooth muscle marker genes ( A ), macrophage marker genes ( B ), and macrophage-related inflammation genes ( C ). All data are averages ± SEM from independent duplicates.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading

    doi: 10.1073/pnas.1735526100

    Figure Lengend Snippet: QRT-PCR determination of SMC and macrophage-related gene expression in SMCs. Subconfluent mouse SMCs were treated with (Chol) or without (Control) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h). In C , control or cholesterol-loaded cells were treated with 30 ng/ml TNF-α for 2 h after cholesterol loading. Total RNA was extracted and subjected to QRT-PCR analysis of smooth muscle marker genes ( A ), macrophage marker genes ( B ), and macrophage-related inflammation genes ( C ). All data are averages ± SEM from independent duplicates.

    Article Snippet: Cholesterol was delivered to cells by using Chol:MβCD complex purchased from Sigma as “water-soluble cholesterol” (catalog no. C4951) containing ≈50 mg of cholesterol/g solid (molar ratio, 1:6 cholesterol/MβCD).

    Techniques: Quantitative RT-PCR, Expressing, Marker

    Cholesterol loading increases the phagocytotic activity of SMCs. ( A ) Subconfluent mouse SMCs were treated with (Chol) or without (Control) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h), followed by incubation with 1-μm latex beads (green) for 20 h. Cells were then washed extensively, fixed, counterstained with 4′,6-diamidino-2-phenylindole (blue, for nuclei) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (red, for cell membrane), and subjected to fluorescent microscopy. (Magnification, ×200.) ( B ) The number of cells and latex beads were also counted to obtain numerical data for phagocytotic activity. All data are averages ± SEM from independent duplicates.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading

    doi: 10.1073/pnas.1735526100

    Figure Lengend Snippet: Cholesterol loading increases the phagocytotic activity of SMCs. ( A ) Subconfluent mouse SMCs were treated with (Chol) or without (Control) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h), followed by incubation with 1-μm latex beads (green) for 20 h. Cells were then washed extensively, fixed, counterstained with 4′,6-diamidino-2-phenylindole (blue, for nuclei) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (red, for cell membrane), and subjected to fluorescent microscopy. (Magnification, ×200.) ( B ) The number of cells and latex beads were also counted to obtain numerical data for phagocytotic activity. All data are averages ± SEM from independent duplicates.

    Article Snippet: Cholesterol was delivered to cells by using Chol:MβCD complex purchased from Sigma as “water-soluble cholesterol” (catalog no. C4951) containing ≈50 mg of cholesterol/g solid (molar ratio, 1:6 cholesterol/MβCD).

    Techniques: Activity Assay, Incubation, Microscopy

    The effects of cholesterol loading on SMC and macrophage-related gene expression in LLC cells ( A ) and NIH/3T3 fibroblasts ( B ). Subconfluent murine LLC cells or NIH/3T3 fibroblasts were treated with (Chol) or without (Control) Chol:MβCD (5 μg/ml) in 0.2% BSA (72 h). Total RNA was extracted and subjected to QRT-PCR analysis. All data are averages ± SEM from independent duplicates. * , α-actin was not detected.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading

    doi: 10.1073/pnas.1735526100

    Figure Lengend Snippet: The effects of cholesterol loading on SMC and macrophage-related gene expression in LLC cells ( A ) and NIH/3T3 fibroblasts ( B ). Subconfluent murine LLC cells or NIH/3T3 fibroblasts were treated with (Chol) or without (Control) Chol:MβCD (5 μg/ml) in 0.2% BSA (72 h). Total RNA was extracted and subjected to QRT-PCR analysis. All data are averages ± SEM from independent duplicates. * , α-actin was not detected.

    Article Snippet: Cholesterol was delivered to cells by using Chol:MβCD complex purchased from Sigma as “water-soluble cholesterol” (catalog no. C4951) containing ≈50 mg of cholesterol/g solid (molar ratio, 1:6 cholesterol/MβCD).

    Techniques: Expressing, Quantitative RT-PCR

    Cholesterol loading leads to smooth muscle foam-cell formation. Subconfluent mouse SMCs were treated with (Chol) or without (Control) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h). Cells were fixed in 10% neutral-buffered formaldehyde and stained with Oil Red O (magnification: A , ×100; Inset , ×400) or harvested to determine cellular cholesterol ( B ) or to extract RNA for QRT-PCR determination of HMG-CoA reductase mRNA levels ( C ). The numerical data are averages ± SEM from independent duplicates.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading

    doi: 10.1073/pnas.1735526100

    Figure Lengend Snippet: Cholesterol loading leads to smooth muscle foam-cell formation. Subconfluent mouse SMCs were treated with (Chol) or without (Control) Chol:MβCD (10 μg/ml) in 0.2% BSA (72 h). Cells were fixed in 10% neutral-buffered formaldehyde and stained with Oil Red O (magnification: A , ×100; Inset , ×400) or harvested to determine cellular cholesterol ( B ) or to extract RNA for QRT-PCR determination of HMG-CoA reductase mRNA levels ( C ). The numerical data are averages ± SEM from independent duplicates.

    Article Snippet: Cholesterol was delivered to cells by using Chol:MβCD complex purchased from Sigma as “water-soluble cholesterol” (catalog no. C4951) containing ≈50 mg of cholesterol/g solid (molar ratio, 1:6 cholesterol/MβCD).

    Techniques: Staining, Quantitative RT-PCR

    Cytotoxicity of Chol:MβCD. Subconfluent mouse SMCs were treated with increasing concentrations of Chol:MβCD in 0.2% BSA for 72 h. Cells were then incubated for 1 h with CellTiter 96 Aqueous One Solution Reagent, convertible to a colored formazan product by metabolically active cells. ( A ) Relative metabolic activity was determined by spectrophotometric measurement (OD = 490 nm) of the accumulation of the formazan product in the medium. ( B ) Total cellular protein was determined by using a Sigma kit. See Materials and Methods for details. Data are averages ± SEM from triplicate wells and repeated at least once.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading

    doi: 10.1073/pnas.1735526100

    Figure Lengend Snippet: Cytotoxicity of Chol:MβCD. Subconfluent mouse SMCs were treated with increasing concentrations of Chol:MβCD in 0.2% BSA for 72 h. Cells were then incubated for 1 h with CellTiter 96 Aqueous One Solution Reagent, convertible to a colored formazan product by metabolically active cells. ( A ) Relative metabolic activity was determined by spectrophotometric measurement (OD = 490 nm) of the accumulation of the formazan product in the medium. ( B ) Total cellular protein was determined by using a Sigma kit. See Materials and Methods for details. Data are averages ± SEM from triplicate wells and repeated at least once.

    Article Snippet: Cholesterol was delivered to cells by using Chol:MβCD complex purchased from Sigma as “water-soluble cholesterol” (catalog no. C4951) containing ≈50 mg of cholesterol/g solid (molar ratio, 1:6 cholesterol/MβCD).

    Techniques: Incubation, Metabolic Labelling, Activity Assay

    The lipid raft disruptor, methyl-β-cyclodextrin (MCD), inhibits TNF-α secretion induced by LPS but does not affect TNF-α secretion induced by cpTi particles with adherent PAMPs. RAW264.7 cells were pre-incubated with the indicated

    Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

    Article Title: Titanium particles activate Toll-like Receptor 4 independently of lipid rafts in RAW264.7 murine macrophages

    doi: 10.1002/jor.21199

    Figure Lengend Snippet: The lipid raft disruptor, methyl-β-cyclodextrin (MCD), inhibits TNF-α secretion induced by LPS but does not affect TNF-α secretion induced by cpTi particles with adherent PAMPs. RAW264.7 cells were pre-incubated with the indicated

    Article Snippet: In selected experiments, RAW264.7 cells were prepared as above and then pre-incubated for 30 minutes with various concentrations of the lipid raft inhibitor methyl-β-cyclodextrin (Sigma) before the addition of either soluble LPS or cpTi particles.

    Techniques: Incubation

    Investigation of the potential N -acylethanolamine molecular targets in N1E-115 cells. N1E-115 cells express cannabinoid receptor CB 1 but not CB 2 , G-protein coupled receptor GPR55, vanilloid receptor TRPV1 and nuclear receptors PPARα and PPARγ ( A ). Detection of mRNA was performed by RT-PCR using mouse brain, spleen and liver as control. The blots are representative of three. Cytotoxicity of AEA (10 µM) ( B ), URB597 (10 µM) ( C ) and AEA + URB597 ( D ) was not significantly affected by CB 1 receptor antagonist (AM251 - 0.1 and 1 µM), TRPV1 receptor antagonist (capsazepine - 10 and 100 nM), PPAR's receptor antagonists (GW6471 and T0070907 - 0.1 and 1 µM) and GPR55 receptor antagonist (cannabidiol, CBD - 0.1 and 1 µM). N1E-115 cells were seeded 5h before treatment (2000 cells/well in microwells) and incubated with AEA alone (10 µM), URB597 alone (10 µM) and combinations of these two molecules. Antagonists were added 1h prior to the addition of AEA and/or URB597. A MTT test was used to evaluate the percentage of viable cells remaining after 72h. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Disruption of lipid rafts inhibits AEA and URB597 mediated effects on N1E-115 cell viability ( E ). Cells were preincubated with methyl-β-cyclodextrin (MCD, 1mM) for 1h prior to the addition of 20 µM of AEA and/or URB597. Cell viability after 72h was assessed with a MTT test. Methyl-β-cyclodextrin had no effect by itself. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Significantly different (**P

    Journal: PLoS ONE

    Article Title: Increasing Antiproliferative Properties of Endocannabinoids in N1E-115 Neuroblastoma Cells through Inhibition of Their Metabolism

    doi: 10.1371/journal.pone.0026823

    Figure Lengend Snippet: Investigation of the potential N -acylethanolamine molecular targets in N1E-115 cells. N1E-115 cells express cannabinoid receptor CB 1 but not CB 2 , G-protein coupled receptor GPR55, vanilloid receptor TRPV1 and nuclear receptors PPARα and PPARγ ( A ). Detection of mRNA was performed by RT-PCR using mouse brain, spleen and liver as control. The blots are representative of three. Cytotoxicity of AEA (10 µM) ( B ), URB597 (10 µM) ( C ) and AEA + URB597 ( D ) was not significantly affected by CB 1 receptor antagonist (AM251 - 0.1 and 1 µM), TRPV1 receptor antagonist (capsazepine - 10 and 100 nM), PPAR's receptor antagonists (GW6471 and T0070907 - 0.1 and 1 µM) and GPR55 receptor antagonist (cannabidiol, CBD - 0.1 and 1 µM). N1E-115 cells were seeded 5h before treatment (2000 cells/well in microwells) and incubated with AEA alone (10 µM), URB597 alone (10 µM) and combinations of these two molecules. Antagonists were added 1h prior to the addition of AEA and/or URB597. A MTT test was used to evaluate the percentage of viable cells remaining after 72h. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Disruption of lipid rafts inhibits AEA and URB597 mediated effects on N1E-115 cell viability ( E ). Cells were preincubated with methyl-β-cyclodextrin (MCD, 1mM) for 1h prior to the addition of 20 µM of AEA and/or URB597. Cell viability after 72h was assessed with a MTT test. Methyl-β-cyclodextrin had no effect by itself. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control. Significantly different (**P

    Article Snippet: All the receptor antagonists (AM251, capsazepine, GW6471, T0070907 and (-)-cannabidiol) were purchased from Tocris Bioscience and the lipid raft disruptor methyl-β-cyclodextrin was from Sigma-Aldrich.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Incubation, MTT Assay

    Effect of macropinocytosis inhibitor treatment on BmNPV infection in Sf21 cells. Sf21 cells were mock-treated or pretreated with different inhibitors (Ehop, Rot, or Lat) for 60 min, then treated with 0.25 mM MβCD or PBS (CTRL) for 30 min and subsequently infected with vBmBac-ph-egfp for 2 h at a MOI of 30. ( A ) Representative images of fluorescence expression in Sf21 cells infected with BmNPV at 72 h p.i. Scale bars, 100 µm. ( B ) Flow cytometry analysis of BmNPV infection in Sf21 cells. (*** p

    Journal: Viruses

    Article Title: Methyl-Beta-Cyclodextrin-Induced Macropinocytosis Results in Increased Infection of Sf21 Cells by Bombyx Mori Nucleopolyhedrovirus

    doi: 10.3390/v11100937

    Figure Lengend Snippet: Effect of macropinocytosis inhibitor treatment on BmNPV infection in Sf21 cells. Sf21 cells were mock-treated or pretreated with different inhibitors (Ehop, Rot, or Lat) for 60 min, then treated with 0.25 mM MβCD or PBS (CTRL) for 30 min and subsequently infected with vBmBac-ph-egfp for 2 h at a MOI of 30. ( A ) Representative images of fluorescence expression in Sf21 cells infected with BmNPV at 72 h p.i. Scale bars, 100 µm. ( B ) Flow cytometry analysis of BmNPV infection in Sf21 cells. (*** p

    Article Snippet: MβCD Treatments and BmNPV Infections MβCD (Sigma-Aldrich, Saint Louis, MO, USA) stock solutions (100 mM) were prepared in phosphate buffer saline (PBS, pH 7.4).

    Techniques: Infection, Fluorescence, Expressing, Flow Cytometry, Cytometry

    MβCD induction post infection rescues BmNPV infection in Sf21 cells. Sf21 cells were mock-treated, or MβCD-treated (0.25 mM) for 30 min before or after vBmBac-ph-egfp infection for 2 h (MOI = 30). ( A ) Representative images of fluorescence expression in Sf21 cells infected with vBmBac-ph-egfp at 72 h p.i. Scale bars, 100 µm. ( B ) Flow cytometry analysis of vBmBac-ph-egfp infection in Sf21 cells at 72 h p.i. (** p

    Journal: Viruses

    Article Title: Methyl-Beta-Cyclodextrin-Induced Macropinocytosis Results in Increased Infection of Sf21 Cells by Bombyx Mori Nucleopolyhedrovirus

    doi: 10.3390/v11100937

    Figure Lengend Snippet: MβCD induction post infection rescues BmNPV infection in Sf21 cells. Sf21 cells were mock-treated, or MβCD-treated (0.25 mM) for 30 min before or after vBmBac-ph-egfp infection for 2 h (MOI = 30). ( A ) Representative images of fluorescence expression in Sf21 cells infected with vBmBac-ph-egfp at 72 h p.i. Scale bars, 100 µm. ( B ) Flow cytometry analysis of vBmBac-ph-egfp infection in Sf21 cells at 72 h p.i. (** p

    Article Snippet: MβCD Treatments and BmNPV Infections MβCD (Sigma-Aldrich, Saint Louis, MO, USA) stock solutions (100 mM) were prepared in phosphate buffer saline (PBS, pH 7.4).

    Techniques: Infection, Fluorescence, Expressing, Flow Cytometry, Cytometry

    The effects of low-dose methyl-beta-cyclodextrin (MβCD) incubation on BmNPV infection in Sf21 cells. ( A ) Representative images of fluorescence or polyhedrin expression in mock-treated or pretreated Sf21 cells infected by BmNPV. Sf21 cells were pretreated with 0.25 mM MβCD or PBS (CTRL) for 30 min and subsequently infected with vBmBac-ph-egfp for 2 h at a multiplicity of infection (MOI) of 30 and observed at 72 h p.i. Red arrows show the occlusion bodies. ( B ) Flow cytometry analysis of BmNPV infectivity in Sf21 cells in the presence of different concentrations of MβCD. Cells were pretreated with the indicated concentrations of MβCD for 30 min and then infected with vBmBac-ph-egfp (MOI = 30). Viral infectivity was measured at 24 h p.i by analyzing the percentage of cells expressing the reporter gene egfp . (** p

    Journal: Viruses

    Article Title: Methyl-Beta-Cyclodextrin-Induced Macropinocytosis Results in Increased Infection of Sf21 Cells by Bombyx Mori Nucleopolyhedrovirus

    doi: 10.3390/v11100937

    Figure Lengend Snippet: The effects of low-dose methyl-beta-cyclodextrin (MβCD) incubation on BmNPV infection in Sf21 cells. ( A ) Representative images of fluorescence or polyhedrin expression in mock-treated or pretreated Sf21 cells infected by BmNPV. Sf21 cells were pretreated with 0.25 mM MβCD or PBS (CTRL) for 30 min and subsequently infected with vBmBac-ph-egfp for 2 h at a multiplicity of infection (MOI) of 30 and observed at 72 h p.i. Red arrows show the occlusion bodies. ( B ) Flow cytometry analysis of BmNPV infectivity in Sf21 cells in the presence of different concentrations of MβCD. Cells were pretreated with the indicated concentrations of MβCD for 30 min and then infected with vBmBac-ph-egfp (MOI = 30). Viral infectivity was measured at 24 h p.i by analyzing the percentage of cells expressing the reporter gene egfp . (** p

    Article Snippet: MβCD Treatments and BmNPV Infections MβCD (Sigma-Aldrich, Saint Louis, MO, USA) stock solutions (100 mM) were prepared in phosphate buffer saline (PBS, pH 7.4).

    Techniques: Incubation, Infection, Fluorescence, Expressing, Flow Cytometry, Cytometry

    Effects of CPZ on BmNPV infection in Sf21 cells. Sf21 cells were treated with CPZ for 30 min and then infected with vBmBac-ph-egfp at a MOI of 50 for 2 h in the mock-treated group or pretreated with 0.25 MβCD for 1 h (no MβCD, pre-MβCD) or post-treated with MβCD after virus infection (post-MβCD). At 72 h p.i., the cells were harvested. ( A ) Representative images of fluorescence expression in Sf21 cells infected with BmNPV. Bars, 100 µm. ( B ) Infection percentage of cells treated with CPZ and MβCD assessed by FCM. ( C ) Relative transcription of lef-3 in CPZ- and MβCD-treated cells. The gene transcription in the 0 µg/mL MβCD treatment condition was set as 1. ( D ) VP39 expression in Sf21 cells treated with increasing concentrations of CPZ in the presence or absence of MβCD using an anti-BmNPV VP39 primary antibody. * p

    Journal: Viruses

    Article Title: Methyl-Beta-Cyclodextrin-Induced Macropinocytosis Results in Increased Infection of Sf21 Cells by Bombyx Mori Nucleopolyhedrovirus

    doi: 10.3390/v11100937

    Figure Lengend Snippet: Effects of CPZ on BmNPV infection in Sf21 cells. Sf21 cells were treated with CPZ for 30 min and then infected with vBmBac-ph-egfp at a MOI of 50 for 2 h in the mock-treated group or pretreated with 0.25 MβCD for 1 h (no MβCD, pre-MβCD) or post-treated with MβCD after virus infection (post-MβCD). At 72 h p.i., the cells were harvested. ( A ) Representative images of fluorescence expression in Sf21 cells infected with BmNPV. Bars, 100 µm. ( B ) Infection percentage of cells treated with CPZ and MβCD assessed by FCM. ( C ) Relative transcription of lef-3 in CPZ- and MβCD-treated cells. The gene transcription in the 0 µg/mL MβCD treatment condition was set as 1. ( D ) VP39 expression in Sf21 cells treated with increasing concentrations of CPZ in the presence or absence of MβCD using an anti-BmNPV VP39 primary antibody. * p

    Article Snippet: MβCD Treatments and BmNPV Infections MβCD (Sigma-Aldrich, Saint Louis, MO, USA) stock solutions (100 mM) were prepared in phosphate buffer saline (PBS, pH 7.4).

    Techniques: Infection, Fluorescence, Expressing

    MβCD treatment induced membrane ruffling, facilitating the BmNPV infection of Sf21 cells. Sf21 cells were treated with or without 0.25 mM MβCD for 30 min and infected with vBmBac-ph-egfp at a MOI of 30 for 2 h at 4 °C. The cells were then fixed and processed for electron microscopy. ( A , D ) scanning electron microscopy analysis on the surface of cells mock-treated or pretreated with MβCD. ( B , E ) transmission electron microscopy analysis of the membrane of cells mock-treated or pretreated with MβCD. Black arrows show the protrusion and closure of ruffles. ( C , F ) Internal subcellular analysis of Sf21 cells mock-treated or pretreated with MβCD. Red arrows show the virions. N, nucleus; PM, plasma membrane; NM, nuclear membrane.

    Journal: Viruses

    Article Title: Methyl-Beta-Cyclodextrin-Induced Macropinocytosis Results in Increased Infection of Sf21 Cells by Bombyx Mori Nucleopolyhedrovirus

    doi: 10.3390/v11100937

    Figure Lengend Snippet: MβCD treatment induced membrane ruffling, facilitating the BmNPV infection of Sf21 cells. Sf21 cells were treated with or without 0.25 mM MβCD for 30 min and infected with vBmBac-ph-egfp at a MOI of 30 for 2 h at 4 °C. The cells were then fixed and processed for electron microscopy. ( A , D ) scanning electron microscopy analysis on the surface of cells mock-treated or pretreated with MβCD. ( B , E ) transmission electron microscopy analysis of the membrane of cells mock-treated or pretreated with MβCD. Black arrows show the protrusion and closure of ruffles. ( C , F ) Internal subcellular analysis of Sf21 cells mock-treated or pretreated with MβCD. Red arrows show the virions. N, nucleus; PM, plasma membrane; NM, nuclear membrane.

    Article Snippet: MβCD Treatments and BmNPV Infections MβCD (Sigma-Aldrich, Saint Louis, MO, USA) stock solutions (100 mM) were prepared in phosphate buffer saline (PBS, pH 7.4).

    Techniques: Infection, Electron Microscopy, Transmission Assay

    Increased cholesterol promotes association of Aβ to neurons. (A,B) Confocal microscopy images of hippocampal neurons exhibiting fluorescence for MAP-2 and demonstrating the distribution of Aβ 42 -FAM aggregates (green) on the cell membrane after treatment with MβCD-Cholesterol complex (200 μM) for 20 min. (A1–A3,B1–B3) ROIs of representative neurites show the overall distribution of Aβ (1 μM, after 1 h) on neuronal processes in more detail. (C,D) Representative traces of primary processes shown in (A,B) , respectively. (E,F) Quantification of Aβ clusters (number and area) in the first 20 μm of hippocampal neuron primary processes showing that both parameters were increased after treatment with MβCD-Cholesterol complex (200 μM) for 20 min. The bars represent the mean ± SEM. Asterisks denote: ∗∗ p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword

    doi: 10.3389/fnagi.2018.00226

    Figure Lengend Snippet: Increased cholesterol promotes association of Aβ to neurons. (A,B) Confocal microscopy images of hippocampal neurons exhibiting fluorescence for MAP-2 and demonstrating the distribution of Aβ 42 -FAM aggregates (green) on the cell membrane after treatment with MβCD-Cholesterol complex (200 μM) for 20 min. (A1–A3,B1–B3) ROIs of representative neurites show the overall distribution of Aβ (1 μM, after 1 h) on neuronal processes in more detail. (C,D) Representative traces of primary processes shown in (A,B) , respectively. (E,F) Quantification of Aβ clusters (number and area) in the first 20 μm of hippocampal neuron primary processes showing that both parameters were increased after treatment with MβCD-Cholesterol complex (200 μM) for 20 min. The bars represent the mean ± SEM. Asterisks denote: ∗∗ p

    Article Snippet: Changes in Cholesterol Levels in the Cell Membrane To increase the levels of cholesterol in the neuron and in the plasma membrane, cells were incubated with media containing MβCD/cholesterol complex (Cholesterol-Water Soluble, Sigma, United States) ( ).

    Techniques: Confocal Microscopy, Fluorescence

    Decreased Aβ association to neurons after lowering cholesterol. (A,B) Confocal microscopy images of hippocampal neurons exhibiting fluorescence for MAP-2 (red) and demonstrating the distribution of Aβ 42 -FAM (green) aggregates on the cell membrane in control and after treatment with MβCD (3 mM) for 30 min. (A1–A3,B1–B3) ROIs of representative neurites showing the overall distribution of Aβ (1 μM, after 1 h treatment) on neuronal processes. (C,D) Representative traces of primary processes shown in (A,B) , respectively. (E,F) Quantification of Aβ clusters (number and area) in the first 20 μm of hippocampal neuron primary processes showing that both parameters decreased after treatment with MβCD (3 mM) for 30 min. The bars represent the mean ± SEM. Asterisks denote: ∗∗ p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword

    doi: 10.3389/fnagi.2018.00226

    Figure Lengend Snippet: Decreased Aβ association to neurons after lowering cholesterol. (A,B) Confocal microscopy images of hippocampal neurons exhibiting fluorescence for MAP-2 (red) and demonstrating the distribution of Aβ 42 -FAM (green) aggregates on the cell membrane in control and after treatment with MβCD (3 mM) for 30 min. (A1–A3,B1–B3) ROIs of representative neurites showing the overall distribution of Aβ (1 μM, after 1 h treatment) on neuronal processes. (C,D) Representative traces of primary processes shown in (A,B) , respectively. (E,F) Quantification of Aβ clusters (number and area) in the first 20 μm of hippocampal neuron primary processes showing that both parameters decreased after treatment with MβCD (3 mM) for 30 min. The bars represent the mean ± SEM. Asterisks denote: ∗∗ p

    Article Snippet: Changes in Cholesterol Levels in the Cell Membrane To increase the levels of cholesterol in the neuron and in the plasma membrane, cells were incubated with media containing MβCD/cholesterol complex (Cholesterol-Water Soluble, Sigma, United States) ( ).

    Techniques: Confocal Microscopy, Fluorescence

    Membrane fluidity changes after decreasing or increasing cholesterol levels. (A) Representative micrographs of hippocampal neurons showing the distribution of scaled GP values in the entire cell and in the membrane area under the experimental conditions previously described. (B) Average GP values showing a decrease in GP in membranes when neurons were treated with MβCD 3 mM. The opposite effect was found when the cells were incubated with 200 μM of soluble cholesterol for 20 min. (C) Coverage analysis showing GP changes in two populations of the membrane domains (GP1 and GP2) for each of the conditions described in (B) In the presence of 3 mM MβCD both domains exhibit lower GP values (more fluid) compared to control conditions, while the addition of cholesterol produce dehydration of both lipid domain populations, becoming more rigid and exhibiting higher GP values. The graphs represent the mean ± SEM. Asterisks denote: ∗∗∗ p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword

    doi: 10.3389/fnagi.2018.00226

    Figure Lengend Snippet: Membrane fluidity changes after decreasing or increasing cholesterol levels. (A) Representative micrographs of hippocampal neurons showing the distribution of scaled GP values in the entire cell and in the membrane area under the experimental conditions previously described. (B) Average GP values showing a decrease in GP in membranes when neurons were treated with MβCD 3 mM. The opposite effect was found when the cells were incubated with 200 μM of soluble cholesterol for 20 min. (C) Coverage analysis showing GP changes in two populations of the membrane domains (GP1 and GP2) for each of the conditions described in (B) In the presence of 3 mM MβCD both domains exhibit lower GP values (more fluid) compared to control conditions, while the addition of cholesterol produce dehydration of both lipid domain populations, becoming more rigid and exhibiting higher GP values. The graphs represent the mean ± SEM. Asterisks denote: ∗∗∗ p

    Article Snippet: Changes in Cholesterol Levels in the Cell Membrane To increase the levels of cholesterol in the neuron and in the plasma membrane, cells were incubated with media containing MβCD/cholesterol complex (Cholesterol-Water Soluble, Sigma, United States) ( ).

    Techniques: Incubation

    Modification of cholesterol levels in hippocampal neurons. (A,B) Quantification of filipin III fluorescence showing relative levels of cholesterol after treatment with MβCD (0.5–3 mM), and after treatment with different concentrations of soluble cholesterol (0–400 μM) in hippocampal neurons. (C,D) Cell viability assay performed after treatments to modify membrane-cholesterol and subsequent Aβ incubation (1 μM for 24 h). The bars represent the mean ± SEM. Asterisks denote: ∗ p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword

    doi: 10.3389/fnagi.2018.00226

    Figure Lengend Snippet: Modification of cholesterol levels in hippocampal neurons. (A,B) Quantification of filipin III fluorescence showing relative levels of cholesterol after treatment with MβCD (0.5–3 mM), and after treatment with different concentrations of soluble cholesterol (0–400 μM) in hippocampal neurons. (C,D) Cell viability assay performed after treatments to modify membrane-cholesterol and subsequent Aβ incubation (1 μM for 24 h). The bars represent the mean ± SEM. Asterisks denote: ∗ p

    Article Snippet: Changes in Cholesterol Levels in the Cell Membrane To increase the levels of cholesterol in the neuron and in the plasma membrane, cells were incubated with media containing MβCD/cholesterol complex (Cholesterol-Water Soluble, Sigma, United States) ( ).

    Techniques: Modification, Fluorescence, Viability Assay, Incubation

    MβCD pretreatment diminishes amplitudes and alters the desensitization of NMDA-induced responses but does not affect kainate-induced responses

    Journal: The Journal of Physiology

    Article Title: Cholesterol modulates open probability and desensitization of NMDA receptors

    doi: 10.1113/jphysiol.2014.288209

    Figure Lengend Snippet: MβCD pretreatment diminishes amplitudes and alters the desensitization of NMDA-induced responses but does not affect kainate-induced responses

    Article Snippet: For cholesterol repletion, DIV 6–7 CGCs were first acutely depleted of cholesterol for 30 min and then subjected to 3.4/20 m m cholesterol/MβCD complex (Sigma).

    Techniques:

    Cholesterol repletion restores the amplitude and desensitization of NMDAR responses in MβCD-pretreated CGCs to control values

    Journal: The Journal of Physiology

    Article Title: Cholesterol modulates open probability and desensitization of NMDA receptors

    doi: 10.1113/jphysiol.2014.288209

    Figure Lengend Snippet: Cholesterol repletion restores the amplitude and desensitization of NMDAR responses in MβCD-pretreated CGCs to control values

    Article Snippet: For cholesterol repletion, DIV 6–7 CGCs were first acutely depleted of cholesterol for 30 min and then subjected to 3.4/20 m m cholesterol/MβCD complex (Sigma).

    Techniques:

    Chronic inhibition of cholesterol biosynthesis by simvastatin induces qualitatively the same effects on NMDA and AMPA/kainate receptors as acute MβCD pretreatment

    Journal: The Journal of Physiology

    Article Title: Cholesterol modulates open probability and desensitization of NMDA receptors

    doi: 10.1113/jphysiol.2014.288209

    Figure Lengend Snippet: Chronic inhibition of cholesterol biosynthesis by simvastatin induces qualitatively the same effects on NMDA and AMPA/kainate receptors as acute MβCD pretreatment

    Article Snippet: For cholesterol repletion, DIV 6–7 CGCs were first acutely depleted of cholesterol for 30 min and then subjected to 3.4/20 m m cholesterol/MβCD complex (Sigma).

    Techniques: Inhibition

    Cholesterol depletion of L. infantum chagasi metacyclic promastigotes by MβCD treatment. (A) Representative full GC outputs of sterols extracted from untreated control metacyclic promastigotes with a load of 2 × 10 5 cell equivalents. The

    Journal: Infection and Immunity

    Article Title: Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

    doi: 10.1128/IAI.00214-13

    Figure Lengend Snippet: Cholesterol depletion of L. infantum chagasi metacyclic promastigotes by MβCD treatment. (A) Representative full GC outputs of sterols extracted from untreated control metacyclic promastigotes with a load of 2 × 10 5 cell equivalents. The

    Article Snippet: Membrane sterols were depleted from stationary-phase or metacyclic promastigotes by incubation of 2 × 108 cells/ml for 1 h in freshly prepared MβCD (Sigma, St. Louis, MO) in RPMI 1640 (GIBCO), ranging in concentration from 0 to 50 mM.

    Techniques:

    C3 Western blotting of MβCD-treated cells versus untreated controls. One hundred nanograms of C3b and iC3b were loaded in lanes 1 and 2, respectively. Metacyclic promastigotes were incubated in a 50% concentration of the indicated sera for 30

    Journal: Infection and Immunity

    Article Title: Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

    doi: 10.1128/IAI.00214-13

    Figure Lengend Snippet: C3 Western blotting of MβCD-treated cells versus untreated controls. One hundred nanograms of C3b and iC3b were loaded in lanes 1 and 2, respectively. Metacyclic promastigotes were incubated in a 50% concentration of the indicated sera for 30

    Article Snippet: Membrane sterols were depleted from stationary-phase or metacyclic promastigotes by incubation of 2 × 108 cells/ml for 1 h in freshly prepared MβCD (Sigma, St. Louis, MO) in RPMI 1640 (GIBCO), ranging in concentration from 0 to 50 mM.

    Techniques: Western Blot, Incubation, Concentration Assay

    Sterol depletion of metacyclic promastigotes affects the outcome of infection. (A and B) Parasite loads in livers and spleens of BALB/c mice infected with metacyclic promastigotes with no treatment (open bars) or treated with 25 mM MβCD (filled

    Journal: Infection and Immunity

    Article Title: Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

    doi: 10.1128/IAI.00214-13

    Figure Lengend Snippet: Sterol depletion of metacyclic promastigotes affects the outcome of infection. (A and B) Parasite loads in livers and spleens of BALB/c mice infected with metacyclic promastigotes with no treatment (open bars) or treated with 25 mM MβCD (filled

    Article Snippet: Membrane sterols were depleted from stationary-phase or metacyclic promastigotes by incubation of 2 × 108 cells/ml for 1 h in freshly prepared MβCD (Sigma, St. Louis, MO) in RPMI 1640 (GIBCO), ranging in concentration from 0 to 50 mM.

    Techniques: Infection, Mouse Assay

    Effect of MβCD on L. infantum chagasi metacyclic promastigote attachment to and survival in macrophages. Human MDMs were infected at an MOI of 5 with opsonized metacyclic promastigotes of untreated control or MβCD-treated (25 mM, 1 h)

    Journal: Infection and Immunity

    Article Title: Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

    doi: 10.1128/IAI.00214-13

    Figure Lengend Snippet: Effect of MβCD on L. infantum chagasi metacyclic promastigote attachment to and survival in macrophages. Human MDMs were infected at an MOI of 5 with opsonized metacyclic promastigotes of untreated control or MβCD-treated (25 mM, 1 h)

    Article Snippet: Membrane sterols were depleted from stationary-phase or metacyclic promastigotes by incubation of 2 × 108 cells/ml for 1 h in freshly prepared MβCD (Sigma, St. Louis, MO) in RPMI 1640 (GIBCO), ranging in concentration from 0 to 50 mM.

    Techniques: Infection

    Released proteins from sterol-depleted promastigotes. (A) Stationary-phase promastigotes were treated with the indicated concentration of MβCD (0 to 10 mM) for 48 h. Filtered supernatants (S) and cells (C) were subjected to SDS-PAGE. MSP was detected

    Journal: Infection and Immunity

    Article Title: Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

    doi: 10.1128/IAI.00214-13

    Figure Lengend Snippet: Released proteins from sterol-depleted promastigotes. (A) Stationary-phase promastigotes were treated with the indicated concentration of MβCD (0 to 10 mM) for 48 h. Filtered supernatants (S) and cells (C) were subjected to SDS-PAGE. MSP was detected

    Article Snippet: Membrane sterols were depleted from stationary-phase or metacyclic promastigotes by incubation of 2 × 108 cells/ml for 1 h in freshly prepared MβCD (Sigma, St. Louis, MO) in RPMI 1640 (GIBCO), ranging in concentration from 0 to 50 mM.

    Techniques: Concentration Assay, SDS Page

    MHV-cell binding after cholesterol extraction or supplementation. HeLa (−) and HeLa-CEACAM (+) cells were cultured in SFM with the indicated doses of MβCD (CD) or cholesterol-MβCD (CHOL) complexes for 30 min at 37°C. Cells were subsequently rinsed and incubated at 4°C for 2 h with 35 S-labeled MHV strain A59 (∼5 × 10 5 cpm). Unadsorbed virions were rinsed away, and cell-associated radioactivity was calculated as a percentage of the total 35 S-labeled virus added.

    Journal: Journal of Virology

    Article Title: Requirements for CEACAMs and Cholesterol during Murine Coronavirus Cell Entry

    doi: 10.1128/JVI.78.6.2682-2692.2004

    Figure Lengend Snippet: MHV-cell binding after cholesterol extraction or supplementation. HeLa (−) and HeLa-CEACAM (+) cells were cultured in SFM with the indicated doses of MβCD (CD) or cholesterol-MβCD (CHOL) complexes for 30 min at 37°C. Cells were subsequently rinsed and incubated at 4°C for 2 h with 35 S-labeled MHV strain A59 (∼5 × 10 5 cpm). Unadsorbed virions were rinsed away, and cell-associated radioactivity was calculated as a percentage of the total 35 S-labeled virus added.

    Article Snippet: methyl -β-Cyclodextrin (MβCD) (Sigma catalog no. C-4555) or cholesterol-MβCD inclusion complexes (Sigma catalog no. C-4951) were serially diluted in serum-free DMEM (SFM), and 1-ml volumes were applied to 106 adherent cells in 10-cm2 dishes at ∼70 to 80% confluency for 30 min at 37°C ( , ).

    Techniques: Binding Assay, Cell Culture, Incubation, Labeling, Radioactivity

    CEACAM-independent fusion after cholesterol extraction. (A) Photos of R18-labeled target cells 3 h after the cells were cocultivated with unlabeled S-bearing (JHM) effector cells. In the rightmost panel, a parallel culture was treated with 5 mM MβCD for 30 min at 37°C and rinsed prior to cocultivation. Bar, 50 μm. (B) Bar graph showing [ 3 H]cholesterol (CHOL) levels in cells (hatched bars) and media (black bars) after exposure to the indicated millimolar concentrations of MβCD (30 min at 37°C). Cells were previously labeled for 18 h with [ 3 H]cholesterol. After CD treatments, media were removed, cells were lysed, and 3 H levels (in disintegrations per minute [DPM]) were determined by scintillation counting. (C) HeLa target cells were treated with Lipofectamine with pTM3-eGFP and then treated with MβCD as described for panel B. Target cells were then overlaid with effector cells presenting S JHM . In this assay, eGFP gene expression is dependent on effector cell-target cell fusion. Micrographs were taken 3 h after cocultivation of effector and target cells. Bar, 100 μm.

    Journal: Journal of Virology

    Article Title: Requirements for CEACAMs and Cholesterol during Murine Coronavirus Cell Entry

    doi: 10.1128/JVI.78.6.2682-2692.2004

    Figure Lengend Snippet: CEACAM-independent fusion after cholesterol extraction. (A) Photos of R18-labeled target cells 3 h after the cells were cocultivated with unlabeled S-bearing (JHM) effector cells. In the rightmost panel, a parallel culture was treated with 5 mM MβCD for 30 min at 37°C and rinsed prior to cocultivation. Bar, 50 μm. (B) Bar graph showing [ 3 H]cholesterol (CHOL) levels in cells (hatched bars) and media (black bars) after exposure to the indicated millimolar concentrations of MβCD (30 min at 37°C). Cells were previously labeled for 18 h with [ 3 H]cholesterol. After CD treatments, media were removed, cells were lysed, and 3 H levels (in disintegrations per minute [DPM]) were determined by scintillation counting. (C) HeLa target cells were treated with Lipofectamine with pTM3-eGFP and then treated with MβCD as described for panel B. Target cells were then overlaid with effector cells presenting S JHM . In this assay, eGFP gene expression is dependent on effector cell-target cell fusion. Micrographs were taken 3 h after cocultivation of effector and target cells. Bar, 100 μm.

    Article Snippet: methyl -β-Cyclodextrin (MβCD) (Sigma catalog no. C-4555) or cholesterol-MβCD inclusion complexes (Sigma catalog no. C-4951) were serially diluted in serum-free DMEM (SFM), and 1-ml volumes were applied to 106 adherent cells in 10-cm2 dishes at ∼70 to 80% confluency for 30 min at 37°C ( , ).

    Techniques: Labeling, Expressing

    Quantification of virus titer on cells pretreated with MβCD or cholesterol-MβCD inclusion complexes. 17 cl 1 cell monolayers were incubated in SFM with the indicated doses of MβCD (A and C) or cholesterol-MβCD complexes (in micrograms per milliliter) (B) for 30 min at 37°C. Cells were subsequently rinsed and overlaid with serial dilutions of the MHV strain JHM, JHM-X, or A59 or with VSV. In one experiment shown in panel A, MβCD was added after JHM adsorption for 30 min. The number of PFU was determined after 3 days. Error bars represent the standard deviations of the means for two independent determinations.

    Journal: Journal of Virology

    Article Title: Requirements for CEACAMs and Cholesterol during Murine Coronavirus Cell Entry

    doi: 10.1128/JVI.78.6.2682-2692.2004

    Figure Lengend Snippet: Quantification of virus titer on cells pretreated with MβCD or cholesterol-MβCD inclusion complexes. 17 cl 1 cell monolayers were incubated in SFM with the indicated doses of MβCD (A and C) or cholesterol-MβCD complexes (in micrograms per milliliter) (B) for 30 min at 37°C. Cells were subsequently rinsed and overlaid with serial dilutions of the MHV strain JHM, JHM-X, or A59 or with VSV. In one experiment shown in panel A, MβCD was added after JHM adsorption for 30 min. The number of PFU was determined after 3 days. Error bars represent the standard deviations of the means for two independent determinations.

    Article Snippet: methyl -β-Cyclodextrin (MβCD) (Sigma catalog no. C-4555) or cholesterol-MβCD inclusion complexes (Sigma catalog no. C-4951) were serially diluted in serum-free DMEM (SFM), and 1-ml volumes were applied to 106 adherent cells in 10-cm2 dishes at ∼70 to 80% confluency for 30 min at 37°C ( , ).

    Techniques: Incubation, Adsorption

    Microscopic depictions (A and B) and quantification (C) of MHV-induced syncytia after cholesterol enrichment or depletion. (A and B) HeLa-CEACAM cells were incubated in SFM containing the indicated concentrations of MβCD (CD) or cholesterol (CHOL) for 30 min at 37°C, rinsed with PBS, incubated for 3 h with MHV strain A59 (MOI, ∼10), and then photographed using phase-contrast (A) or fluorescence (B) microscopy. In panel B, cells were labeled with the fluorescent cytosolic dye CMFDA. Bars, 200 μm. (C) The indicated doses of CD and cholesterol (chol) were incubated with HeLa-CEACAM cells that had been prepared for cell fusion-dependent reporter gene activation, as described in Materials and Methods. Cells were rinsed with PBS, A59 virions were applied (MOI, ∼10), and reporter gene product β-galactosidase enzyme activities in lysates were quantified by measuring CPRG substrate turnover at an optical density of 590 nm (OD 590).

    Journal: Journal of Virology

    Article Title: Requirements for CEACAMs and Cholesterol during Murine Coronavirus Cell Entry

    doi: 10.1128/JVI.78.6.2682-2692.2004

    Figure Lengend Snippet: Microscopic depictions (A and B) and quantification (C) of MHV-induced syncytia after cholesterol enrichment or depletion. (A and B) HeLa-CEACAM cells were incubated in SFM containing the indicated concentrations of MβCD (CD) or cholesterol (CHOL) for 30 min at 37°C, rinsed with PBS, incubated for 3 h with MHV strain A59 (MOI, ∼10), and then photographed using phase-contrast (A) or fluorescence (B) microscopy. In panel B, cells were labeled with the fluorescent cytosolic dye CMFDA. Bars, 200 μm. (C) The indicated doses of CD and cholesterol (chol) were incubated with HeLa-CEACAM cells that had been prepared for cell fusion-dependent reporter gene activation, as described in Materials and Methods. Cells were rinsed with PBS, A59 virions were applied (MOI, ∼10), and reporter gene product β-galactosidase enzyme activities in lysates were quantified by measuring CPRG substrate turnover at an optical density of 590 nm (OD 590).

    Article Snippet: methyl -β-Cyclodextrin (MβCD) (Sigma catalog no. C-4555) or cholesterol-MβCD inclusion complexes (Sigma catalog no. C-4951) were serially diluted in serum-free DMEM (SFM), and 1-ml volumes were applied to 106 adherent cells in 10-cm2 dishes at ∼70 to 80% confluency for 30 min at 37°C ( , ).

    Techniques: Incubation, Fluorescence, Microscopy, Labeling, Activation Assay

    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

    NADPH oxidase assembly and activity in a reconstituted cell-free system depends on cholesterol. ( A ) Control or mβCD-treated purified macrophage membranes were extracted in TX-100 or Brij-58, and distribution of cyt b 558 subunits in the detergent-insoluble pellet (P) and soluble fraction (S) was determined by Western blotting. ( B ) Superoxide production of control or mβCD-extracted purified macrophage membranes in the reconstituted amphiphile-activated system as measured by cytochrome c reduction. Results are expressed as percent cytochrome c reduction of control, and represent mean and s.d. of four independent experiments. Superoxide production of control membranes corresponded to 15.9 mol superoxide/mol cyt b 558 heme/s. ( C ) Control or mβCD-extracted membranes mixed with cytosolic subunits were pelleted, and association of p67phox, p47phox, and Rac1 with membrane determined by lysis in Laemmli buffer and Western blotting. ( D ) Distribution of cytosolic subunits in detergent-insoluble pellet (P) and soluble fraction (S) after extraction of control membranes in Brij-58.

    Journal: The EMBO Journal

    Article Title: The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly

    doi: 10.1038/sj.emboj.7600066

    Figure Lengend Snippet: NADPH oxidase assembly and activity in a reconstituted cell-free system depends on cholesterol. ( A ) Control or mβCD-treated purified macrophage membranes were extracted in TX-100 or Brij-58, and distribution of cyt b 558 subunits in the detergent-insoluble pellet (P) and soluble fraction (S) was determined by Western blotting. ( B ) Superoxide production of control or mβCD-extracted purified macrophage membranes in the reconstituted amphiphile-activated system as measured by cytochrome c reduction. Results are expressed as percent cytochrome c reduction of control, and represent mean and s.d. of four independent experiments. Superoxide production of control membranes corresponded to 15.9 mol superoxide/mol cyt b 558 heme/s. ( C ) Control or mβCD-extracted membranes mixed with cytosolic subunits were pelleted, and association of p67phox, p47phox, and Rac1 with membrane determined by lysis in Laemmli buffer and Western blotting. ( D ) Distribution of cytosolic subunits in detergent-insoluble pellet (P) and soluble fraction (S) after extraction of control membranes in Brij-58.

    Article Snippet: TX100, Brij-58, Brij-98, mβCD, and cholesterol-saturated mβCD were purchased from Sigma.

    Techniques: Activity Assay, Purification, Western Blot, Lysis

    NADPH oxidase activity in Ra2 microglia and HL60 cells requires cholesterol. ( A–D ) Ra2 control (•) or cholesterol-extracted cells (○) were stimulated with (A) fMLP, (B) PMA, or (C) IgG-opsonized zymosan particles, and superoxide production measured continuously by luminol-enhanced chemiluminescence. Bar graph (D) shows mean and s.d. of three independent experiments carried out as in (A–C). Superoxide production of control cells was assigned an arbitrary value of 100. ( E–H ) Control (•), mβCD-extracted (○), or mβCD-extracted and then cholesterol-reconstituted (▵) HL60 cells were stimulated with either (E) PMA, (F) ionomycin, or (G) fMLP. Subsequently, superoxide production was measured as above. Bar graph (H) shows mean and s.d. of at least three independent experiments, expressed as percent superoxide production of control cells. Empty bars represent cholesterol-extracted cells, and filled bars represent cholesterol-replenished cells.

    Journal: The EMBO Journal

    Article Title: The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly

    doi: 10.1038/sj.emboj.7600066

    Figure Lengend Snippet: NADPH oxidase activity in Ra2 microglia and HL60 cells requires cholesterol. ( A–D ) Ra2 control (•) or cholesterol-extracted cells (○) were stimulated with (A) fMLP, (B) PMA, or (C) IgG-opsonized zymosan particles, and superoxide production measured continuously by luminol-enhanced chemiluminescence. Bar graph (D) shows mean and s.d. of three independent experiments carried out as in (A–C). Superoxide production of control cells was assigned an arbitrary value of 100. ( E–H ) Control (•), mβCD-extracted (○), or mβCD-extracted and then cholesterol-reconstituted (▵) HL60 cells were stimulated with either (E) PMA, (F) ionomycin, or (G) fMLP. Subsequently, superoxide production was measured as above. Bar graph (H) shows mean and s.d. of at least three independent experiments, expressed as percent superoxide production of control cells. Empty bars represent cholesterol-extracted cells, and filled bars represent cholesterol-replenished cells.

    Article Snippet: TX100, Brij-58, Brij-98, mβCD, and cholesterol-saturated mβCD were purchased from Sigma.

    Techniques: Activity Assay

    P47phox and p67phox segregation to low-density detergent (Brij-58) resistant membrane is increased by cell activation, and their membrane translocation is cholesterol-dependent. ( A ) Brij-58 lysates of control or PMA-stimulated HL60 cells were centrifuged to equilibrium in a sucrose gradient, and then analyzed by Western blotting using antibodies to p67phox, p47phox, or Rac1. ( B ) Control or cholesterol-extracted HL60 cells were stimulated with PMA, and subsequently the particulate membrane fraction was analyzed for the presence of p67phox, p47phox, or Rac1 by Western blotting. Equal aliquots were also analyzed for p22phox as loading control. ( C ) HL60 cells were subjected to cholesterol extraction (mβCD), in some cases followed by cholesterol replenishment (mβCD chol), before stimulation with PMA. p67phox, p47phox, Rac1, and p22phox was analyzed by Western blotting as described above. ( D ) The graph represents mean and s.e. of three independent experiments performed as in (C). Individual p67phox, p47phox, and Rac1 bands were quantitated densitometrically, and the results expressed as percent translocation in cholesterol-extracted (white bars) or cholesterol-replenished (filled bars) cells relative to control cells. ( E ) HL60 cells stimulated with PMA were subjected to chemical crosslinking with DTSP and the particulate membrane fraction, purified as above, was subjected to sucrose gradient centrifugation. Collected fractions were electrophoresed under reducing conditions and analyzed by Western blotting with anti-p67phox, p47phox, Rac1, gp91phox, and p22phox antibodies.

    Journal: The EMBO Journal

    Article Title: The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly

    doi: 10.1038/sj.emboj.7600066

    Figure Lengend Snippet: P47phox and p67phox segregation to low-density detergent (Brij-58) resistant membrane is increased by cell activation, and their membrane translocation is cholesterol-dependent. ( A ) Brij-58 lysates of control or PMA-stimulated HL60 cells were centrifuged to equilibrium in a sucrose gradient, and then analyzed by Western blotting using antibodies to p67phox, p47phox, or Rac1. ( B ) Control or cholesterol-extracted HL60 cells were stimulated with PMA, and subsequently the particulate membrane fraction was analyzed for the presence of p67phox, p47phox, or Rac1 by Western blotting. Equal aliquots were also analyzed for p22phox as loading control. ( C ) HL60 cells were subjected to cholesterol extraction (mβCD), in some cases followed by cholesterol replenishment (mβCD chol), before stimulation with PMA. p67phox, p47phox, Rac1, and p22phox was analyzed by Western blotting as described above. ( D ) The graph represents mean and s.e. of three independent experiments performed as in (C). Individual p67phox, p47phox, and Rac1 bands were quantitated densitometrically, and the results expressed as percent translocation in cholesterol-extracted (white bars) or cholesterol-replenished (filled bars) cells relative to control cells. ( E ) HL60 cells stimulated with PMA were subjected to chemical crosslinking with DTSP and the particulate membrane fraction, purified as above, was subjected to sucrose gradient centrifugation. Collected fractions were electrophoresed under reducing conditions and analyzed by Western blotting with anti-p67phox, p47phox, Rac1, gp91phox, and p22phox antibodies.

    Article Snippet: TX100, Brij-58, Brij-98, mβCD, and cholesterol-saturated mβCD were purchased from Sigma.

    Techniques: Activation Assay, Translocation Assay, Western Blot, Purification, Gradient Centrifugation

    Gp91phox and p22phox segregate to the buoyant low-density detergent (Brij-58, Brij-98) resistant membrane fraction. Ra2 microglia ( A–H ) or HL60 ( I–Q ) cells were lysed in Brij-58 (A–D, I–M) or Brij-98 (E–H, N–Q), in some cases with prior mβCD-facilitated cholesterol extraction (B, F, J), and the lysate was centrifuged to equilibrium in a 35–15% sucrose gradient. Recovered fractions (no. 12 is the bottom, high-density fraction of the gradient) and the solubilized pellet (P) were analyzed by Western blotting using antibodies against gp91phox (gp91), p22phox (p22), transferrin receptor (TfnR), β1-integrin (β1), flotillin-2 (Flo-2), or Lyn as indicated.

    Journal: The EMBO Journal

    Article Title: The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly

    doi: 10.1038/sj.emboj.7600066

    Figure Lengend Snippet: Gp91phox and p22phox segregate to the buoyant low-density detergent (Brij-58, Brij-98) resistant membrane fraction. Ra2 microglia ( A–H ) or HL60 ( I–Q ) cells were lysed in Brij-58 (A–D, I–M) or Brij-98 (E–H, N–Q), in some cases with prior mβCD-facilitated cholesterol extraction (B, F, J), and the lysate was centrifuged to equilibrium in a 35–15% sucrose gradient. Recovered fractions (no. 12 is the bottom, high-density fraction of the gradient) and the solubilized pellet (P) were analyzed by Western blotting using antibodies against gp91phox (gp91), p22phox (p22), transferrin receptor (TfnR), β1-integrin (β1), flotillin-2 (Flo-2), or Lyn as indicated.

    Article Snippet: TX100, Brij-58, Brij-98, mβCD, and cholesterol-saturated mβCD were purchased from Sigma.

    Techniques: Western Blot

    PKC membrane translocation is inhibited after mβCD-mediated cholesterol extraction. ( A ) Brij-58 lysates of control or PMA-stimulated HL60 cells were centrifuged to equilibrium in a sucrose gradient, and then analyzed by Western blotting using antibodies to PKCβ. ( B ) HL60 cells were subjected to cholesterol extraction (mβCD), in some cases followed by cholesterol replenishment (mβCD chol), before stimulation with PMA, and translocation of PKCβ to the particulate membrane fraction analyzed by Western blotting. Equal aliquots were also analyzed for p22phox as loading control. ( C ) The graph represents mean and s.e. of three independent experiments performed as in (B). The results are expressed as percent translocation in cholesterol-extracted (empty bars) or cholesterol-replenished (filled bars) cells relative to control cells.

    Journal: The EMBO Journal

    Article Title: The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly

    doi: 10.1038/sj.emboj.7600066

    Figure Lengend Snippet: PKC membrane translocation is inhibited after mβCD-mediated cholesterol extraction. ( A ) Brij-58 lysates of control or PMA-stimulated HL60 cells were centrifuged to equilibrium in a sucrose gradient, and then analyzed by Western blotting using antibodies to PKCβ. ( B ) HL60 cells were subjected to cholesterol extraction (mβCD), in some cases followed by cholesterol replenishment (mβCD chol), before stimulation with PMA, and translocation of PKCβ to the particulate membrane fraction analyzed by Western blotting. Equal aliquots were also analyzed for p22phox as loading control. ( C ) The graph represents mean and s.e. of three independent experiments performed as in (B). The results are expressed as percent translocation in cholesterol-extracted (empty bars) or cholesterol-replenished (filled bars) cells relative to control cells.

    Article Snippet: TX100, Brij-58, Brij-98, mβCD, and cholesterol-saturated mβCD were purchased from Sigma.

    Techniques: Translocation Assay, Western Blot

    Dose-dependent impairment of PMNL shear-responses by membrane-modifying cholesterol:MβCD conjugates correlates with their effects on cell membrane fluidity. A : PMNL shear-responses (PRR; %) were determined for cells pretreated with 0–10

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Membrane cholesterol modulates the fluid shear stress response of polymorphonuclear leukocytes via its effects on membrane fluidity

    doi: 10.1152/ajpcell.00458.2010

    Figure Lengend Snippet: Dose-dependent impairment of PMNL shear-responses by membrane-modifying cholesterol:MβCD conjugates correlates with their effects on cell membrane fluidity. A : PMNL shear-responses (PRR; %) were determined for cells pretreated with 0–10

    Article Snippet: Migrating leukocytes were allowed to adhere to plasma proteins adsorbed on glass substrates (Fisher Scientific) in Plasma-Lyte buffer (containing 2.5 mM CaCl2 ) for 10 min and subsequently incubated with either 2 mM MβCD (Sigma-Aldrich), 5 μg/ml filipin (Sigma-Aldrich), or 2 μg/ml cholesterol:MβCD complexes (Sigma-Aldrich) for 15 min. After these treatments, migrating cells were washed with excess Plasma-Lyte containing 2.5 mM CaCl2 and exposed to shear stress in the presence of 0–15 mM BnOH (Acros Organics).

    Techniques:

    Macrophages release particles enriched in cholesterol. ( A ) Macrophages were loaded with cholesterol/MβCD. SEM and nanoSIMS images of the macrophage after a short incubation with [ 15 N]ALO-D4. The SEM image shows a lawn of particles outside the macrophage; the nanoSIMS image (scaled at two different settings) reveals binding of [ 15 N]ALO-D4 to the particles, indicating that they contain accessible cholesterol. The boxed regions are shown below at higher magnification, again showing binding of [ 15 N]ALO-D4 to macrophage-derived particles. (Scale bars, 5 μm.) The bar graph shows 15 N/ 14 N levels for the cell body and particles of two cells (60 particles were quantified). The y axis starts at 0.0037, the natural abundance of 15 N. Data are shown as mean ± SD. ( B ) Macrophages were loaded with acetyl-LDL. Particles released by macrophages that had been loaded with acetyl-LDL (50 μg/mL) (yellow arrows) are visible in secondary electron (SE) and 12 C 14 N − nanoSIMS images. Composite 12 C 14 N − or secondary electron (SE) (gray) and 15 N/ 14 N ratio (red) images show binding of [ 15 N]ALO-D4 to particles. (Scale bars, 2 μm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Macrophages release plasma membrane-derived particles rich in accessible cholesterol

    doi: 10.1073/pnas.1810724115

    Figure Lengend Snippet: Macrophages release particles enriched in cholesterol. ( A ) Macrophages were loaded with cholesterol/MβCD. SEM and nanoSIMS images of the macrophage after a short incubation with [ 15 N]ALO-D4. The SEM image shows a lawn of particles outside the macrophage; the nanoSIMS image (scaled at two different settings) reveals binding of [ 15 N]ALO-D4 to the particles, indicating that they contain accessible cholesterol. The boxed regions are shown below at higher magnification, again showing binding of [ 15 N]ALO-D4 to macrophage-derived particles. (Scale bars, 5 μm.) The bar graph shows 15 N/ 14 N levels for the cell body and particles of two cells (60 particles were quantified). The y axis starts at 0.0037, the natural abundance of 15 N. Data are shown as mean ± SD. ( B ) Macrophages were loaded with acetyl-LDL. Particles released by macrophages that had been loaded with acetyl-LDL (50 μg/mL) (yellow arrows) are visible in secondary electron (SE) and 12 C 14 N − nanoSIMS images. Composite 12 C 14 N − or secondary electron (SE) (gray) and 15 N/ 14 N ratio (red) images show binding of [ 15 N]ALO-D4 to particles. (Scale bars, 2 μm.)

    Article Snippet: RAW 267.4 cells were loaded with unlabeled cholesterol/MβCD complexes (Sigma).

    Techniques: Incubation, Binding Assay, Derivative Assay

    Surface expression of HA-CB1F238L compared with HA-CB1wt after inhibition of clathrin coated pit or caveolae mediated endocytosis. (A) HEK293 cells stably expressing HA-CB1wt or HA-CB1F238L were treated either for 15 min with 20 μM PitStop2™ to inhibit clathrin coated pit endocytosis or for 30 min with 5 mM methyl-β-cyclo-dextrin (MβCD) to inhibit caveolae mediated endocytosis. Surface expression was analyzed by a trypsin protection assay. (B) Only MβCD treatment significantly increased surface expression of CB1F238L receptor. (Two way ANOVA and Bonferroni’s post hoc test. Data are presented as the mean ± SEM of n = 4 independent experiments. Surface HA-CB1 was calculated and expressed as percent of total HA-CB1 as described in “Materials and Methods” section * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The F238L Point Mutation in the Cannabinoid Type 1 Receptor Enhances Basal Endocytosis via Lipid Rafts

    doi: 10.3389/fnmol.2018.00230

    Figure Lengend Snippet: Surface expression of HA-CB1F238L compared with HA-CB1wt after inhibition of clathrin coated pit or caveolae mediated endocytosis. (A) HEK293 cells stably expressing HA-CB1wt or HA-CB1F238L were treated either for 15 min with 20 μM PitStop2™ to inhibit clathrin coated pit endocytosis or for 30 min with 5 mM methyl-β-cyclo-dextrin (MβCD) to inhibit caveolae mediated endocytosis. Surface expression was analyzed by a trypsin protection assay. (B) Only MβCD treatment significantly increased surface expression of CB1F238L receptor. (Two way ANOVA and Bonferroni’s post hoc test. Data are presented as the mean ± SEM of n = 4 independent experiments. Surface HA-CB1 was calculated and expressed as percent of total HA-CB1 as described in “Materials and Methods” section * p

    Article Snippet: For endocytosis experiments, cells were treated either with 100 μM WIN-55212-2 (BN0544, BioTrend Chemicals AG) or 100 μM SR141716 (generously provided by the NIMH Chemical Synthesis and Drug Supply Program) in SFM for 45 min, with 20 μM PitStop2™ (Abcam) for 15 min or 5 mM methyl-β-cyclo-dextrin (MβCD; Sigma) in SFM for 30 min. WIN-55212-2, SR141716 and PitStop2™were dissolved in DMSO, MβCD was dissolved in SFM.

    Techniques: Expressing, Inhibition, Stable Transfection