cytochalasin d  (Millipore)


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    Structured Review

    Millipore cytochalasin d
    Functional validation of the differential coordination between Arp2/3 and VASP in strong accelerating protrusion. a , b t-SNE plots of the denoised protrusion velocity time series of the whole sample overlaid with the density of data. c , d t-SNE plots of the denoised velocities of the sub-clusters (Cluster III-1 and III-2) in Cluster III. e , f Comparison of the proportion of Cluster III-1 and III-2 upon <t>Cytochalasin</t> D treatment ( e ) or CK666 treatment ( f ). The error bars indicate 95% confidence interval of the mean of the cluster proportions. * p
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 142 article reviews
    Price from $9.99 to $1999.99
    cytochalasin d - by Bioz Stars, 2020-02
    99/100 stars

    Images

    1) Product Images from "Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging"

    Article Title: Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04030-0

    Functional validation of the differential coordination between Arp2/3 and VASP in strong accelerating protrusion. a , b t-SNE plots of the denoised protrusion velocity time series of the whole sample overlaid with the density of data. c , d t-SNE plots of the denoised velocities of the sub-clusters (Cluster III-1 and III-2) in Cluster III. e , f Comparison of the proportion of Cluster III-1 and III-2 upon Cytochalasin D treatment ( e ) or CK666 treatment ( f ). The error bars indicate 95% confidence interval of the mean of the cluster proportions. * p
    Figure Legend Snippet: Functional validation of the differential coordination between Arp2/3 and VASP in strong accelerating protrusion. a , b t-SNE plots of the denoised protrusion velocity time series of the whole sample overlaid with the density of data. c , d t-SNE plots of the denoised velocities of the sub-clusters (Cluster III-1 and III-2) in Cluster III. e , f Comparison of the proportion of Cluster III-1 and III-2 upon Cytochalasin D treatment ( e ) or CK666 treatment ( f ). The error bars indicate 95% confidence interval of the mean of the cluster proportions. * p

    Techniques Used: Functional Assay

    2) Product Images from "The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii"

    Article Title: The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002007

    Parasites lacking RON8 are deficient in invasion likely through increased detachment from host cells. A ) Quantification of invasion using red/green assays demonstrates a substantial invasion defect for Δron8 parasites that is rescued upon complementation. Green bars represent internal/penetrated parasites, while red bars depict attached/extracellular parasites for wildtype, Δron8 , or R8c strains allowed to invade fibroblast monolayers for 1 hour. For each strain, at least 250 total parasites were counted from nine random fields per sample, and values are presented as internal (Int) or external (Ext) parasites per field. Data are mean values +/− SEM (error bars) for two independent experiments performed in triplicate. The asterisk indicates that parasite penetration is significantly lower (p value = 0.0245 using a Student's two-tailed t-test) in Δ ron8 parasites compared to wildtype. B ) Initial stages of attachment are unaffected in Δ ron8 parasites. Equal numbers of wildtype, Δron8 , or R8c parasites were preincubated with 1 µM cytochalasin D for 15 min prior to incubation with host fibroblasts in the presence of cytochalasin D, then fixed and stained in detergent-free conditions with rabbit antisera against SAG1. For each strain, values are displayed as total numbers of parasites counted divided over nine random fields. The data is expressed as mean values +/− SEM for two independent experiments performed in triplicate.
    Figure Legend Snippet: Parasites lacking RON8 are deficient in invasion likely through increased detachment from host cells. A ) Quantification of invasion using red/green assays demonstrates a substantial invasion defect for Δron8 parasites that is rescued upon complementation. Green bars represent internal/penetrated parasites, while red bars depict attached/extracellular parasites for wildtype, Δron8 , or R8c strains allowed to invade fibroblast monolayers for 1 hour. For each strain, at least 250 total parasites were counted from nine random fields per sample, and values are presented as internal (Int) or external (Ext) parasites per field. Data are mean values +/− SEM (error bars) for two independent experiments performed in triplicate. The asterisk indicates that parasite penetration is significantly lower (p value = 0.0245 using a Student's two-tailed t-test) in Δ ron8 parasites compared to wildtype. B ) Initial stages of attachment are unaffected in Δ ron8 parasites. Equal numbers of wildtype, Δron8 , or R8c parasites were preincubated with 1 µM cytochalasin D for 15 min prior to incubation with host fibroblasts in the presence of cytochalasin D, then fixed and stained in detergent-free conditions with rabbit antisera against SAG1. For each strain, values are displayed as total numbers of parasites counted divided over nine random fields. The data is expressed as mean values +/− SEM for two independent experiments performed in triplicate.

    Techniques Used: Significance Assay, Two Tailed Test, Incubation, Staining

    3) Product Images from "Helicobacter pylori cag Pathogenicity Island (cagPAI) Involved in Bacterial Internalization and IL-8 Induced Responses via NOD1- and MyD88-Dependent Mechanisms in Human Biliary Epithelial Cells"

    Article Title: Helicobacter pylori cag Pathogenicity Island (cagPAI) Involved in Bacterial Internalization and IL-8 Induced Responses via NOD1- and MyD88-Dependent Mechanisms in Human Biliary Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077358

    Actin polymerization and H. pylori internalization. A . Effect of cytochalasin D (actin polymerization inhibitor) on H. pylori internalization in biliary (KKU-100 and KKU-M156) and gastric cells (AGS) . After treatment with cytochalasin D, cells were incubated with H. pylori wild type, cagA or cag PAI mutant strains for 6 h. The H. pylori internalization was assessed by bacterial culture. The percentage of H. pylori internalization in cytochalasin D-treated cells was compared to the number of H. pylori internalization in untreated control cells . B . Effect of α5β1 integrin antibodies on H. pylori internalization in biliary (KKU-100 and KKU-M156) and gastric (AGS) cells . After pre-treatment with α5β1 integrin antibodies, cells were incubated with H. pylori wild type, cagA - or cag PAI - strains for 6 h. H. pylori internalization was accessed by bacterial culture. The percentage of H. pylori internalization in α5β1 integrin-antibody-treated cells was compared to the number of H. pylori internalization in untreated control cells. Data are the mean ± SEM of triplicate experiments. * p
    Figure Legend Snippet: Actin polymerization and H. pylori internalization. A . Effect of cytochalasin D (actin polymerization inhibitor) on H. pylori internalization in biliary (KKU-100 and KKU-M156) and gastric cells (AGS) . After treatment with cytochalasin D, cells were incubated with H. pylori wild type, cagA or cag PAI mutant strains for 6 h. The H. pylori internalization was assessed by bacterial culture. The percentage of H. pylori internalization in cytochalasin D-treated cells was compared to the number of H. pylori internalization in untreated control cells . B . Effect of α5β1 integrin antibodies on H. pylori internalization in biliary (KKU-100 and KKU-M156) and gastric (AGS) cells . After pre-treatment with α5β1 integrin antibodies, cells were incubated with H. pylori wild type, cagA - or cag PAI - strains for 6 h. H. pylori internalization was accessed by bacterial culture. The percentage of H. pylori internalization in α5β1 integrin-antibody-treated cells was compared to the number of H. pylori internalization in untreated control cells. Data are the mean ± SEM of triplicate experiments. * p

    Techniques Used: Incubation, Mutagenesis

    4) Product Images from "Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFN?-Induced STAT1 Transcriptional Activity"

    Article Title: Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFN?-Induced STAT1 Transcriptional Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060215

    Toxoplasma induces STAT1 phosphorylation and nuclear translocation in BMDC. (A) BMDC were left in medium alone (Med), infected with type I (RH), II (PTG) or III (M774.1) strains of Toxoplasma (3∶1 ratio of parasites to cells), or treated with murine IFNγ (100 ng/ml), prior to fractionation into cytoplasmic (C) and nuclear (N) extracts at the time points indicated. Samples were subjected to immunoblot analysis for phospho-Tyr701-STAT1 (pY-STAT1) and phospho-Ser-STAT1 (pS-STAT1). PARP and Rab5a served as loading controls for nuclear and cytoplasmic fractions, respectively. (B) Cells were infected with live parasites of the three strains as in (A) or exposed to heat-inactivated (HI) tachyzoites for six hours. Cytoplasmic and nuclear fractions were collected and immunoblot analyis was performed as in (A). (C) RH parasites were pre-treated for 10 min on ice with 1 μM cytochalasin D (CytD) prior to infection in the continued presence of the drug. Cells treated with the solvent DMSO alone served as controls. Cells were fractionated after six hours and subjected to immunoblot analysis for pY-STAT1. (D and E) BMDC were treated with IFNγ or infected with RH in comparison with either the cps1-1 replication-deficient strain (D) or the ΔROP16 strain (E). Samples were fractionated after 6 and 20 hours and subjected to immunoblot analysis for pY-STAT1. All experiments were repeated at least three times with similar results.
    Figure Legend Snippet: Toxoplasma induces STAT1 phosphorylation and nuclear translocation in BMDC. (A) BMDC were left in medium alone (Med), infected with type I (RH), II (PTG) or III (M774.1) strains of Toxoplasma (3∶1 ratio of parasites to cells), or treated with murine IFNγ (100 ng/ml), prior to fractionation into cytoplasmic (C) and nuclear (N) extracts at the time points indicated. Samples were subjected to immunoblot analysis for phospho-Tyr701-STAT1 (pY-STAT1) and phospho-Ser-STAT1 (pS-STAT1). PARP and Rab5a served as loading controls for nuclear and cytoplasmic fractions, respectively. (B) Cells were infected with live parasites of the three strains as in (A) or exposed to heat-inactivated (HI) tachyzoites for six hours. Cytoplasmic and nuclear fractions were collected and immunoblot analyis was performed as in (A). (C) RH parasites were pre-treated for 10 min on ice with 1 μM cytochalasin D (CytD) prior to infection in the continued presence of the drug. Cells treated with the solvent DMSO alone served as controls. Cells were fractionated after six hours and subjected to immunoblot analysis for pY-STAT1. (D and E) BMDC were treated with IFNγ or infected with RH in comparison with either the cps1-1 replication-deficient strain (D) or the ΔROP16 strain (E). Samples were fractionated after 6 and 20 hours and subjected to immunoblot analysis for pY-STAT1. All experiments were repeated at least three times with similar results.

    Techniques Used: Translocation Assay, Infection, Fractionation

    5) Product Images from "Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages"

    Article Title: Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages

    Journal: Infection and Immunity

    doi:

    Fragmentation of genomic DNA from about 5 × 10 6 to 1 × 10 7 J774 macrophages infected with MC1061/pYMZ80 at an MOI of 100 with or without cytochalasin D (Cyt-D) at 12 h p.i. DNA purification and gel electrophoresis were carried out as described in the text. MC1061/pYMZ80-infected cells generated ladders of multimers of 180 bp characteristic of apoptosis. Lanes: 1, 100-bp DNA ladder; 2, vector control strain MC1061/pUC18; 3, ClyA-expressing strain MC1061/pYMZ80; 4, ClyA-expressing strain MC1061/pYMZ80 with 1 μg of Cyt-D ml −1 .
    Figure Legend Snippet: Fragmentation of genomic DNA from about 5 × 10 6 to 1 × 10 7 J774 macrophages infected with MC1061/pYMZ80 at an MOI of 100 with or without cytochalasin D (Cyt-D) at 12 h p.i. DNA purification and gel electrophoresis were carried out as described in the text. MC1061/pYMZ80-infected cells generated ladders of multimers of 180 bp characteristic of apoptosis. Lanes: 1, 100-bp DNA ladder; 2, vector control strain MC1061/pUC18; 3, ClyA-expressing strain MC1061/pYMZ80; 4, ClyA-expressing strain MC1061/pYMZ80 with 1 μg of Cyt-D ml −1 .

    Techniques Used: Infection, DNA Purification, Nucleic Acid Electrophoresis, Generated, Plasmid Preparation, Expressing

    6) Product Images from "CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells"

    Article Title: CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-06238-4

    CD56 mediated fungal recognition is dependent on actin and CD56 blocking inhibits NK cell function. NK cells were incubated with ( a ) cytochalasin D (Cyt. D, n = 4), ( b ) colchicine (Col., n = 3) and ( c ) CD56 blocking antibody (bAb, n = 4) prior to co-cultivation with the fungus (AF GT). Percentage of CD56 positive cells was determined after an incubation with the fungus for 0, 3, 6, and 9 h. A paired student’s t-test was performed to compare NK + ctrl + AF GT against NK + CytD + AF GT or NK + Col + AF GT ( a , b ). Control samples were cultured in the presence of the corresponding control solution. Percentage of CD56 and CD69 positive cells was assessed after a co-cultivation for 9 h. CD56 and CD69 expression was analysed using flow cytometry. NK cells were defined as NKp46 + CD3 − . Supernatants derived from CD56 blocking experiments were analysed by ProcartaPlex TM multiplex immunoassays (n = 4, d – f ). The concentration of ( d ) MIP-1α, ( e ) MIP-1β and ( f ) RANTES detectable in supernatants is displayed in pg/ml. A paired student’s t-test was performed to compare ( c ) NK + AF GT against NK + bAb + AF GT and ( d – f ) NK against + AF GT and + AF GT against + bAb AF GT. Data are represented as mean ± SEM. Significant differences are indicated by an asterisk (*p
    Figure Legend Snippet: CD56 mediated fungal recognition is dependent on actin and CD56 blocking inhibits NK cell function. NK cells were incubated with ( a ) cytochalasin D (Cyt. D, n = 4), ( b ) colchicine (Col., n = 3) and ( c ) CD56 blocking antibody (bAb, n = 4) prior to co-cultivation with the fungus (AF GT). Percentage of CD56 positive cells was determined after an incubation with the fungus for 0, 3, 6, and 9 h. A paired student’s t-test was performed to compare NK + ctrl + AF GT against NK + CytD + AF GT or NK + Col + AF GT ( a , b ). Control samples were cultured in the presence of the corresponding control solution. Percentage of CD56 and CD69 positive cells was assessed after a co-cultivation for 9 h. CD56 and CD69 expression was analysed using flow cytometry. NK cells were defined as NKp46 + CD3 − . Supernatants derived from CD56 blocking experiments were analysed by ProcartaPlex TM multiplex immunoassays (n = 4, d – f ). The concentration of ( d ) MIP-1α, ( e ) MIP-1β and ( f ) RANTES detectable in supernatants is displayed in pg/ml. A paired student’s t-test was performed to compare ( c ) NK + AF GT against NK + bAb + AF GT and ( d – f ) NK against + AF GT and + AF GT against + bAb AF GT. Data are represented as mean ± SEM. Significant differences are indicated by an asterisk (*p

    Techniques Used: Blocking Assay, Cell Function Assay, Incubation, Cell Culture, Expressing, Flow Cytometry, Cytometry, Derivative Assay, Multiplex Assay, Concentration Assay

    7) Product Images from "Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging"

    Article Title: Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04030-0

    Functional validation of the differential coordination between Arp2/3 and VASP in strong accelerating protrusion. a , b t-SNE plots of the denoised protrusion velocity time series of the whole sample overlaid with the density of data. c , d t-SNE plots of the denoised velocities of the sub-clusters (Cluster III-1 and III-2) in Cluster III. e , f Comparison of the proportion of Cluster III-1 and III-2 upon Cytochalasin D treatment ( e ) or CK666 treatment ( f ). The error bars indicate 95% confidence interval of the mean of the cluster proportions. * p
    Figure Legend Snippet: Functional validation of the differential coordination between Arp2/3 and VASP in strong accelerating protrusion. a , b t-SNE plots of the denoised protrusion velocity time series of the whole sample overlaid with the density of data. c , d t-SNE plots of the denoised velocities of the sub-clusters (Cluster III-1 and III-2) in Cluster III. e , f Comparison of the proportion of Cluster III-1 and III-2 upon Cytochalasin D treatment ( e ) or CK666 treatment ( f ). The error bars indicate 95% confidence interval of the mean of the cluster proportions. * p

    Techniques Used: Functional Assay

    8) Product Images from "Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells"

    Article Title: Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells

    Journal: Infection and Immunity

    doi:

    Invasion of MAC-T cells by S. aureus Novel. A dose response invasion assay was performed by exposing MAC-T cell monolayers to various concentrations of S. aureus cells so that the MOI was altered within the range indicated. Culture media were supplemented with either gentamicin alone (open squares) or gentamicin plus cytochalasin D (solid squares). Additional controls showed that overnight incubation of S. aureus cells in the presence of 0.5 μg of cytochalasin D per ml resulted in no loss in viability (data not shown). Data are from a representative experiment repeated four times. Error bars represent the means ± the standard errors of the means.
    Figure Legend Snippet: Invasion of MAC-T cells by S. aureus Novel. A dose response invasion assay was performed by exposing MAC-T cell monolayers to various concentrations of S. aureus cells so that the MOI was altered within the range indicated. Culture media were supplemented with either gentamicin alone (open squares) or gentamicin plus cytochalasin D (solid squares). Additional controls showed that overnight incubation of S. aureus cells in the presence of 0.5 μg of cytochalasin D per ml resulted in no loss in viability (data not shown). Data are from a representative experiment repeated four times. Error bars represent the means ± the standard errors of the means.

    Techniques Used: Invasion Assay, Incubation

    Related Articles

    Centrifugation:

    Article Title: Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFN?-Induced STAT1 Transcriptional Activity
    Article Snippet: In vitro Infections and Stimuli Infection of BMDCs was accomplished through addition of tachyzoites to cell cultures at a ratio of 3∶1 (parasites:BMDCs) followed by brief centrifugation (200×g, 3 min) to initiate contact between cells and parasites. .. For cytochalasin D experiments, BMDC were pretreated for 10 min at 4°C with cytochalasin D (Calbiochem) at a final concentration of 1 μM or with the solvent DMSO (Sigma) alone.

    Filtration:

    Article Title: Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages
    Article Snippet: Proteins were diluted in complete medium and sterilized by filtration through a 0.22-μm-pore-size membrane (Schleicher & Schuell FD 030/3). .. For testing the effect of cytochalasin D ( ) on cytotoxicity, cells were pretreated with 1 μg of cytochalasin D (Sigma) ml−1 for 30 min before bacterial infection, and cytochalasin D was maintained throughout the experiment.

    Cytometry:

    Article Title: CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells
    Article Snippet: Cytochalasin D and colchicine treatment NK cells were treated with 10 μM cytochalasin D (Sigma), 10 μM colchicine or the perspective DMSO and ethanole control for 30 min at 37 °C. .. Expression of CD56 was determined using flow cytometry.

    Blocking Assay:

    Article Title: Mycobacterium tuberculosis Hip1 Dampens Macrophage Proinflammatory Responses by Limiting Toll-Like Receptor 2 Activation ▿
    Article Snippet: .. For phagosome blocking experiments using cytochalasin D, macrophages were treated with 10 μM cytochalasin D (Sigma-Aldrich, St. Louis, MO) for 1 h before infection. .. Macrophages were infected in triplicate with H37Rv or the hip1 mutant as described above.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Mycobacterium tuberculosis Hip1 Dampens Macrophage Proinflammatory Responses by Limiting Toll-Like Receptor 2 Activation ▿
    Article Snippet: Cell-free supernatants from macrophage monolayers were isolated at various time points and assayed for cytokines by enzyme-linked immunosorbent assay (ELISA), using duo set kits for IL-1β (BD Biosciences, San Diego, CA), TNF-α and IL-6 (R & D Systems, Minneapolis, MN), and IL-18 (MBL International Corporation, Woburn, MA). .. For phagosome blocking experiments using cytochalasin D, macrophages were treated with 10 μM cytochalasin D (Sigma-Aldrich, St. Louis, MO) for 1 h before infection.

    Incubation:

    Article Title: Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging
    Article Snippet: .. For Cytochalasin D experiments, cells were incubated with DMSO or Cytochalasin D (Sigma) for half an hour before imaging. .. All microscopy was performed using the set up as follows: Nikon Ti-E inverted motorized microscope (including motorized focus, objective nosepiece, fluorescence filter turret, and condenser turret) with integrated Perfect Focus System, Nikon Plan Apo 1.4 NA DIC optics (60×), Yokogawa CSU-X1 spinning disk confocal head with manual emission filter wheel with Spectral Applied Research Borealis modification, Spectral Applied Research custom laser merge module (LMM-7) with AOTF and solid state 445 nm (200 mW), 488 nm (200 mW), 514 nm (150 mW), 561 nm (200 mW), and 637 nm (140 mW) lasers, Semrock 405/488/561/647 and 442/514/647 dichroic mirrors, Ludl encoded XY stage, Ludl piezo Z sample holder for high speed optical sectioning, Prior fast transmitted and epi-fluorescence light path shutters, Hamamatsu Flash 4.0 LT sCMOS camera, 37 °C microscope incubator enclosure with 5% CO2 delivery (In Vivo), Molecular Devices MetaMorph v7.7, TMC vibration-isolation table.

    Article Title: Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells
    Article Snippet: .. To test the inhibition of S. aureus invasion by cytochalasin D, experiments were carried out as described for the invasion assays except that prior to inoculation with bacteria, MAC-T cell monolayers were incubated at 37°C with 7% CO2 for 2 h with invasion medium containing 0.5 μg of cytochalasin D (Sigma) per ml. .. The occurrence of apoptosis in MAC-T cells was evaluated by three different methods: (i) DNA laddering, (ii) a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay ( , , , ), and (iii) microscopic observation of morphological changes in the cells.

    Article Title: The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii
    Article Snippet: After quenching as above, the samples were blocked in PBS/3%BSA for 25 minutes and incubated with rabbit anti-SAG1 diluted in PBS/3%BSA for 1 hour, then washed five times in 1X PBS and permeabilized with PBT buffer for 30 minutes prior to incubation with mouse anti-ROP7 diluted in PBT buffer as a second primary step. .. Attachment and evacuole assays utilizing cytochalasin D were performed as described ; wildtype, Δron8 , and R8c parasites were scrape-syringed as above and resuspended in Endo buffer (44.7 mM K2 SO4 , 10 mM Mg2 SO4 , 106 mM sucrose, 5 mM glucose, 20 mM Tris, 0.35% wt/vol BSA, pH 8.2) containing 1 µM cytochalasin D (Sigma-Aldrich).

    Article Title: Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface ?1 integrins
    Article Snippet: .. For experiments with cytochalasin D, cells were incubated with Fluo-4 and ligand-coated beads for 10 min, and then exposed to cytochalasin D (2 μg/mL, Sigma) in the presence of Fluo-4 for 20 min prior to washing. .. Change in the fluorescence intensity at a single wavelength (488 nm excitation) in response to force was measured using a Nikon Eclipse TE-2000-E Fluorescence microscope equipped with a 1000 W High Pressure Mercury Burner (Olympus Optical Company).

    Cell Culture:

    Article Title: Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging
    Article Snippet: Paragraph title: Cell culture and drug treatment ... For Cytochalasin D experiments, cells were incubated with DMSO or Cytochalasin D (Sigma) for half an hour before imaging.

    Article Title: CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells
    Article Snippet: Cytochalasin D and colchicine treatment NK cells were treated with 10 μM cytochalasin D (Sigma), 10 μM colchicine or the perspective DMSO and ethanole control for 30 min at 37 °C. .. NK cells were cultured alone or with A . fumigatus germ tubes (MOI 0.5) for 0, 3, 6 and 9 h in the presence of 5 μM cytochalasin D or colchicine or the perspective DMSO or ethanol controls.

    Article Title: Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages
    Article Snippet: Paragraph title: Cytotoxic effects of purified ClyA protein and ClyA-expressing E. coli on fresh or cultured human and murine monocytes/macrophages. ... For testing the effect of cytochalasin D ( ) on cytotoxicity, cells were pretreated with 1 μg of cytochalasin D (Sigma) ml−1 for 30 min before bacterial infection, and cytochalasin D was maintained throughout the experiment.

    Expressing:

    Article Title: Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging
    Article Snippet: GFP-Arp3 expressing PtK1 cells were further selected by G418 before imaging. .. For Cytochalasin D experiments, cells were incubated with DMSO or Cytochalasin D (Sigma) for half an hour before imaging.

    Article Title: CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells
    Article Snippet: Cytochalasin D and colchicine treatment NK cells were treated with 10 μM cytochalasin D (Sigma), 10 μM colchicine or the perspective DMSO and ethanole control for 30 min at 37 °C. .. Expression of CD56 was determined using flow cytometry.

    Article Title: The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii
    Article Snippet: To examine whether the Δron8 parasites could be rescued by host cells exogenously expressing RON8, we used a competition growth assay in cells with and without RON8 expression. .. Attachment and evacuole assays utilizing cytochalasin D were performed as described ; wildtype, Δron8 , and R8c parasites were scrape-syringed as above and resuspended in Endo buffer (44.7 mM K2 SO4 , 10 mM Mg2 SO4 , 106 mM sucrose, 5 mM glucose, 20 mM Tris, 0.35% wt/vol BSA, pH 8.2) containing 1 µM cytochalasin D (Sigma-Aldrich).

    Flow Cytometry:

    Article Title: CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells
    Article Snippet: Cytochalasin D and colchicine treatment NK cells were treated with 10 μM cytochalasin D (Sigma), 10 μM colchicine or the perspective DMSO and ethanole control for 30 min at 37 °C. .. Expression of CD56 was determined using flow cytometry.

    Infection:

    Article Title: Mycobacterium tuberculosis Hip1 Dampens Macrophage Proinflammatory Responses by Limiting Toll-Like Receptor 2 Activation ▿
    Article Snippet: .. For phagosome blocking experiments using cytochalasin D, macrophages were treated with 10 μM cytochalasin D (Sigma-Aldrich, St. Louis, MO) for 1 h before infection. .. Macrophages were infected in triplicate with H37Rv or the hip1 mutant as described above.

    Article Title: Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages
    Article Snippet: .. For testing the effect of cytochalasin D ( ) on cytotoxicity, cells were pretreated with 1 μg of cytochalasin D (Sigma) ml−1 for 30 min before bacterial infection, and cytochalasin D was maintained throughout the experiment. .. Treatment of the bacteria and eukaryotic cells with cytochalasin D at the above concentration did not significantly reduce cell or bacterial viability (data not shown).

    Article Title: Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFN?-Induced STAT1 Transcriptional Activity
    Article Snippet: In vitro Infections and Stimuli Infection of BMDCs was accomplished through addition of tachyzoites to cell cultures at a ratio of 3∶1 (parasites:BMDCs) followed by brief centrifugation (200×g, 3 min) to initiate contact between cells and parasites. .. For cytochalasin D experiments, BMDC were pretreated for 10 min at 4°C with cytochalasin D (Calbiochem) at a final concentration of 1 μM or with the solvent DMSO (Sigma) alone.

    Inhibition:

    Article Title: Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging
    Article Snippet: For Arp2/3 inhibition experiments, cells were incubated with 50 μM of CK666 or CK689 (EMD Millipore) for an hour before imaging. .. For Cytochalasin D experiments, cells were incubated with DMSO or Cytochalasin D (Sigma) for half an hour before imaging.

    Article Title: Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells
    Article Snippet: .. To test the inhibition of S. aureus invasion by cytochalasin D, experiments were carried out as described for the invasion assays except that prior to inoculation with bacteria, MAC-T cell monolayers were incubated at 37°C with 7% CO2 for 2 h with invasion medium containing 0.5 μg of cytochalasin D (Sigma) per ml. .. The occurrence of apoptosis in MAC-T cells was evaluated by three different methods: (i) DNA laddering, (ii) a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay ( , , , ), and (iii) microscopic observation of morphological changes in the cells.

    Article Title: Helicobacter pylori cag Pathogenicity Island (cagPAI) Involved in Bacterial Internalization and IL-8 Induced Responses via NOD1- and MyD88-Dependent Mechanisms in Human Biliary Epithelial Cells
    Article Snippet: .. Inhibition of bacterial internalization by cytochalasin D or α5β1 integrin antibodies Cells were grown in 12-well tissue culture plates and pre-treated for 30 min with either cytochalasin D (5 μg/ml) (Sigma, St. Louis, MO) or α5β1 integrin antibodies (5 μg/ml) (AIIB2 rat anti-human β1 integrin, IgG1, BIIG2 rat anti-human α5 integrin, IgG2b κ integrin-blocking antibodies, Developmental Studies Hybridoma Bank, University of Iowa, USA) for 1 h at 37°C with 5% CO2 , as previously described [ ]. ..

    Imaging:

    Article Title: Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging
    Article Snippet: .. For Cytochalasin D experiments, cells were incubated with DMSO or Cytochalasin D (Sigma) for half an hour before imaging. .. All microscopy was performed using the set up as follows: Nikon Ti-E inverted motorized microscope (including motorized focus, objective nosepiece, fluorescence filter turret, and condenser turret) with integrated Perfect Focus System, Nikon Plan Apo 1.4 NA DIC optics (60×), Yokogawa CSU-X1 spinning disk confocal head with manual emission filter wheel with Spectral Applied Research Borealis modification, Spectral Applied Research custom laser merge module (LMM-7) with AOTF and solid state 445 nm (200 mW), 488 nm (200 mW), 514 nm (150 mW), 561 nm (200 mW), and 637 nm (140 mW) lasers, Semrock 405/488/561/647 and 442/514/647 dichroic mirrors, Ludl encoded XY stage, Ludl piezo Z sample holder for high speed optical sectioning, Prior fast transmitted and epi-fluorescence light path shutters, Hamamatsu Flash 4.0 LT sCMOS camera, 37 °C microscope incubator enclosure with 5% CO2 delivery (In Vivo), Molecular Devices MetaMorph v7.7, TMC vibration-isolation table.

    Article Title: Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface ?1 integrins
    Article Snippet: For experiments with cytochalasin D, cells were incubated with Fluo-4 and ligand-coated beads for 10 min, and then exposed to cytochalasin D (2 μg/mL, Sigma) in the presence of Fluo-4 for 20 min prior to washing. .. Results are presented as F/Fo, where F is the average fluorescence intensity in the ROI at each time point during time lapse imaging, and Fo is the fluorescence intensity in the ROI at time=0.

    Recombinant:

    Article Title: Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFN?-Induced STAT1 Transcriptional Activity
    Article Snippet: In other experiments, cells were treated with recombinant murine IFNγ (100 ng/mL, Peprotech) or first pre-infected with tachyzoites for two hours followed by IFNγ treatment. .. For cytochalasin D experiments, BMDC were pretreated for 10 min at 4°C with cytochalasin D (Calbiochem) at a final concentration of 1 μM or with the solvent DMSO (Sigma) alone.

    Immunofluorescence:

    Article Title: The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii
    Article Snippet: Paragraph title: Immunofluorescence and invasion assays ... Attachment and evacuole assays utilizing cytochalasin D were performed as described ; wildtype, Δron8 , and R8c parasites were scrape-syringed as above and resuspended in Endo buffer (44.7 mM K2 SO4 , 10 mM Mg2 SO4 , 106 mM sucrose, 5 mM glucose, 20 mM Tris, 0.35% wt/vol BSA, pH 8.2) containing 1 µM cytochalasin D (Sigma-Aldrich).

    Invasion Assay:

    Article Title: Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells
    Article Snippet: Paragraph title: Invasion assay. ... To test the inhibition of S. aureus invasion by cytochalasin D, experiments were carried out as described for the invasion assays except that prior to inoculation with bacteria, MAC-T cell monolayers were incubated at 37°C with 7% CO2 for 2 h with invasion medium containing 0.5 μg of cytochalasin D (Sigma) per ml.

    Fluorescence:

    Article Title: Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface ?1 integrins
    Article Snippet: For experiments with cytochalasin D, cells were incubated with Fluo-4 and ligand-coated beads for 10 min, and then exposed to cytochalasin D (2 μg/mL, Sigma) in the presence of Fluo-4 for 20 min prior to washing. .. Change in the fluorescence intensity at a single wavelength (488 nm excitation) in response to force was measured using a Nikon Eclipse TE-2000-E Fluorescence microscope equipped with a 1000 W High Pressure Mercury Burner (Olympus Optical Company).

    Isolation:

    Article Title: Mycobacterium tuberculosis Hip1 Dampens Macrophage Proinflammatory Responses by Limiting Toll-Like Receptor 2 Activation ▿
    Article Snippet: Cell-free supernatants from macrophage monolayers were isolated at various time points and assayed for cytokines by enzyme-linked immunosorbent assay (ELISA), using duo set kits for IL-1β (BD Biosciences, San Diego, CA), TNF-α and IL-6 (R & D Systems, Minneapolis, MN), and IL-18 (MBL International Corporation, Woburn, MA). .. For phagosome blocking experiments using cytochalasin D, macrophages were treated with 10 μM cytochalasin D (Sigma-Aldrich, St. Louis, MO) for 1 h before infection.

    Article Title: Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages
    Article Snippet: Human polymorphonuclear leukocytes and monocytes were isolated following a standardized procedure as described previously ( , ). .. For testing the effect of cytochalasin D ( ) on cytotoxicity, cells were pretreated with 1 μg of cytochalasin D (Sigma) ml−1 for 30 min before bacterial infection, and cytochalasin D was maintained throughout the experiment.

    Growth Assay:

    Article Title: The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii
    Article Snippet: To examine whether the Δron8 parasites could be rescued by host cells exogenously expressing RON8, we used a competition growth assay in cells with and without RON8 expression. .. Attachment and evacuole assays utilizing cytochalasin D were performed as described ; wildtype, Δron8 , and R8c parasites were scrape-syringed as above and resuspended in Endo buffer (44.7 mM K2 SO4 , 10 mM Mg2 SO4 , 106 mM sucrose, 5 mM glucose, 20 mM Tris, 0.35% wt/vol BSA, pH 8.2) containing 1 µM cytochalasin D (Sigma-Aldrich).

    Labeling:

    Article Title: The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii
    Article Snippet: Attachment and evacuole assays utilizing cytochalasin D were performed as described ; wildtype, Δron8 , and R8c parasites were scrape-syringed as above and resuspended in Endo buffer (44.7 mM K2 SO4 , 10 mM Mg2 SO4 , 106 mM sucrose, 5 mM glucose, 20 mM Tris, 0.35% wt/vol BSA, pH 8.2) containing 1 µM cytochalasin D (Sigma-Aldrich). .. Media was replaced with prewarmed DMEM/10% FBS containing 1 µM cytochalasin D and incubation continued for another 15 min at 37°C before fixation with formaldehyde and immunofluorescence/counting as described above for red/green invasion assays (except that instead of anti-ROP7 antibody, evacuoles were labeled with monoclonal anti-ROP2/3/4 antibody).

    Purification:

    Article Title: Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages
    Article Snippet: Paragraph title: Cytotoxic effects of purified ClyA protein and ClyA-expressing E. coli on fresh or cultured human and murine monocytes/macrophages. ... For testing the effect of cytochalasin D ( ) on cytotoxicity, cells were pretreated with 1 μg of cytochalasin D (Sigma) ml−1 for 30 min before bacterial infection, and cytochalasin D was maintained throughout the experiment.

    Microscopy:

    Article Title: Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface ?1 integrins
    Article Snippet: For experiments with cytochalasin D, cells were incubated with Fluo-4 and ligand-coated beads for 10 min, and then exposed to cytochalasin D (2 μg/mL, Sigma) in the presence of Fluo-4 for 20 min prior to washing. .. Change in the fluorescence intensity at a single wavelength (488 nm excitation) in response to force was measured using a Nikon Eclipse TE-2000-E Fluorescence microscope equipped with a 1000 W High Pressure Mercury Burner (Olympus Optical Company).

    Irradiation:

    Article Title: Mycobacterium tuberculosis Hip1 Dampens Macrophage Proinflammatory Responses by Limiting Toll-Like Receptor 2 Activation ▿
    Article Snippet: Alternatively, macrophages were infected with heat-killed or irradiated M. tuberculosis at an MOI of 10 or 200 μg, respectively, in DMEM/F-12 medium containing 5% LCM. .. For phagosome blocking experiments using cytochalasin D, macrophages were treated with 10 μM cytochalasin D (Sigma-Aldrich, St. Louis, MO) for 1 h before infection.

    Multiplex Assay:

    Article Title: Mycobacterium tuberculosis Hip1 Dampens Macrophage Proinflammatory Responses by Limiting Toll-Like Receptor 2 Activation ▿
    Article Snippet: Multiplex ELISAs were carried out using a Luminex multiplex kit and were analyzed using a Bio-Plex 200 system (Millipore, Billerica, MA). .. For phagosome blocking experiments using cytochalasin D, macrophages were treated with 10 μM cytochalasin D (Sigma-Aldrich, St. Louis, MO) for 1 h before infection.

    In Vitro:

    Article Title: Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFN?-Induced STAT1 Transcriptional Activity
    Article Snippet: Paragraph title: In vitro Infections and Stimuli ... For cytochalasin D experiments, BMDC were pretreated for 10 min at 4°C with cytochalasin D (Calbiochem) at a final concentration of 1 μM or with the solvent DMSO (Sigma) alone.

    Laser Capture Microdissection:

    Article Title: Mycobacterium tuberculosis Hip1 Dampens Macrophage Proinflammatory Responses by Limiting Toll-Like Receptor 2 Activation ▿
    Article Snippet: Alternatively, macrophages were infected with heat-killed or irradiated M. tuberculosis at an MOI of 10 or 200 μg, respectively, in DMEM/F-12 medium containing 5% LCM. .. For phagosome blocking experiments using cytochalasin D, macrophages were treated with 10 μM cytochalasin D (Sigma-Aldrich, St. Louis, MO) for 1 h before infection.

    Knock-Out:

    Article Title: The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii
    Article Snippet: No difference in the rate at which the wildtype parasites outcompeted the knockout was observed, indicating that exogenously expressed RON8 cannot complement the invasion defect. .. Attachment and evacuole assays utilizing cytochalasin D were performed as described ; wildtype, Δron8 , and R8c parasites were scrape-syringed as above and resuspended in Endo buffer (44.7 mM K2 SO4 , 10 mM Mg2 SO4 , 106 mM sucrose, 5 mM glucose, 20 mM Tris, 0.35% wt/vol BSA, pH 8.2) containing 1 µM cytochalasin D (Sigma-Aldrich).

    Concentration Assay:

    Article Title: Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages
    Article Snippet: For testing the effect of cytochalasin D ( ) on cytotoxicity, cells were pretreated with 1 μg of cytochalasin D (Sigma) ml−1 for 30 min before bacterial infection, and cytochalasin D was maintained throughout the experiment. .. Treatment of the bacteria and eukaryotic cells with cytochalasin D at the above concentration did not significantly reduce cell or bacterial viability (data not shown).

    Article Title: Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFN?-Induced STAT1 Transcriptional Activity
    Article Snippet: .. For cytochalasin D experiments, BMDC were pretreated for 10 min at 4°C with cytochalasin D (Calbiochem) at a final concentration of 1 μM or with the solvent DMSO (Sigma) alone. .. Cells were then infected with tachyzoites or treated with recombinant murine IFNγ for 6 hours in the continued presence of the drug.

    Staining:

    Article Title: Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging
    Article Snippet: #150682.) for 2 days and stained with 5 μg ml−1 CellMask Deep Red (Invitrogen) following manufacturer’s protocol. .. For Cytochalasin D experiments, cells were incubated with DMSO or Cytochalasin D (Sigma) for half an hour before imaging.

    Article Title: The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii
    Article Snippet: Parasites staining with both anti-SAG1 and ROP7 denoted attached but uninvaded parasites, while those staining only for ROP7 were scored as internalized. .. Attachment and evacuole assays utilizing cytochalasin D were performed as described ; wildtype, Δron8 , and R8c parasites were scrape-syringed as above and resuspended in Endo buffer (44.7 mM K2 SO4 , 10 mM Mg2 SO4 , 106 mM sucrose, 5 mM glucose, 20 mM Tris, 0.35% wt/vol BSA, pH 8.2) containing 1 µM cytochalasin D (Sigma-Aldrich).

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    Millipore cytochalasin d
    Elastic modulus of CHO cells treated with nocodazole, trypsin or cytochalasin D. p-values were derived from Microsoft Excel’s Student’s T-test. N=10.
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Millipore actin cytoskeleton inhibitor cytochalasin d
    Signaling events, the F-actin <t>cytoskeleton,</t> and lipid rafts are involved in F1845-induced PS externalization on PLB-985 cells. Before challenge with the different E. coli strains, PLB-985 cells were pretreated for 1 h with several modulating agents, including the tyrosine kinase inhibitor genistein, the lipid raft-modifying drug MβCD, the actin cytoskeleton inhibitor cytochalasin D, and the protein kinase C inhibitor staurosporine. At the end of the incubation period the cells were stained with both annexin V-PE and 7-AAD, and the percentages of 7-AAD-negative, annexin V-positive cells were determined by flow cytometry. The data are data from five experiments and are means ± standard errors of the means. *, P
    Actin Cytoskeleton Inhibitor Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/actin cytoskeleton inhibitor cytochalasin d/product/Millipore
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    79
    Millipore actin filament disruptor cytochalasin d
    Effects of actin manipulation on oocyte effective tension. (A) Phalloidin staining of a GVI oocyte (left) and a metaphase II (MII) egg (right). Polarity develops during meiotic maturation, such that the metaphase II egg has an actin-rich cap over the meiotic spindle. Red, actin; blue, DNA. Scale bar, (left) 20 μm; (right) 22 μm. (B) Effects on effective tension (T eff ) of actin manipulation by <t>cytochalasin</t> D treatment of GVI oocytes (*p
    Actin Filament Disruptor Cytochalasin D, 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
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    Image Search Results


    Elastic modulus of CHO cells treated with nocodazole, trypsin or cytochalasin D. p-values were derived from Microsoft Excel’s Student’s T-test. N=10.

    Journal: Methods (San Diego, Calif.)

    Article Title: Rearrangement of microtubule network under biochemical and mechanical stimulations

    doi: 10.1016/j.ymeth.2013.02.014

    Figure Lengend Snippet: Elastic modulus of CHO cells treated with nocodazole, trypsin or cytochalasin D. p-values were derived from Microsoft Excel’s Student’s T-test. N=10.

    Article Snippet: Nocodazole (Calbiochem) and cytochalasin D (Calbiochem) were added to the cell medium to achieve final concentrations of 20 μM and 5 μM, respectively.

    Techniques: Derivative Assay

    Cytoskeleton polymerization may affect the rate of FM1-43 accumulation in activated T cells. Activated T cells were stained with Oregon Green phalloidin after fixation. Before fixation, cells were incubated in normal Tyrode solution (A), in the presence of 1 μ M thapsigargin (5 min, B), 10 μ M cytochalasin D (20 min, C), 200 nM calyculin A (20 min, D), or both 10 μ M cytochalasin D and calyculin A (20 min, E). Pretreatments with cytochalasin D and calyculin A were done at 37 °C for 20 min whereas thapsigargin was applied at 24 °C. In parallel experiments, activated T cells were exposed to FM1-43 for 30 s (F, H, J, L), or 21 min (G, I, K, M) in normal Tyrode solution (F, G); after pretreatment with 10 μ M cytochalasin D (H, I); or 200 nM calyculin A (J, K); or both 10 μ M cytochalasin D and 200 nM calyculin A (L, M). Note that in panels J and K a bright FM1-43 fluorescence spot that appears to be located in the cytosol (cell on the left) is in the extracellular space, because staining emerged immediately after dye application. This area was excluded from the analysis. The time courses of FM1-43 accumulation were acquired at 24 °C. Scale bars are 10 μ m. (N) Time courses of FM1-43 accumulation in normal Tyrode solution (closed circles); in the presence of 10 μ M cytochalasin D (open triangles); in the presence of 200 nM calyculin A (open squares); and in the presence of both 10 μ M cytochalasin D and 200 nM calyculin A (closed triangles). Each time course is an average of six experiments from different cultures.

    Journal: Experimental cell research

    Article Title: Regulation of membrane trafficking and subcellular organization of endocytic compartments revealed with FM1-43 in resting and activated human T cells

    doi:

    Figure Lengend Snippet: Cytoskeleton polymerization may affect the rate of FM1-43 accumulation in activated T cells. Activated T cells were stained with Oregon Green phalloidin after fixation. Before fixation, cells were incubated in normal Tyrode solution (A), in the presence of 1 μ M thapsigargin (5 min, B), 10 μ M cytochalasin D (20 min, C), 200 nM calyculin A (20 min, D), or both 10 μ M cytochalasin D and calyculin A (20 min, E). Pretreatments with cytochalasin D and calyculin A were done at 37 °C for 20 min whereas thapsigargin was applied at 24 °C. In parallel experiments, activated T cells were exposed to FM1-43 for 30 s (F, H, J, L), or 21 min (G, I, K, M) in normal Tyrode solution (F, G); after pretreatment with 10 μ M cytochalasin D (H, I); or 200 nM calyculin A (J, K); or both 10 μ M cytochalasin D and 200 nM calyculin A (L, M). Note that in panels J and K a bright FM1-43 fluorescence spot that appears to be located in the cytosol (cell on the left) is in the extracellular space, because staining emerged immediately after dye application. This area was excluded from the analysis. The time courses of FM1-43 accumulation were acquired at 24 °C. Scale bars are 10 μ m. (N) Time courses of FM1-43 accumulation in normal Tyrode solution (closed circles); in the presence of 10 μ M cytochalasin D (open triangles); in the presence of 200 nM calyculin A (open squares); and in the presence of both 10 μ M cytochalasin D and 200 nM calyculin A (closed triangles). Each time course is an average of six experiments from different cultures.

    Article Snippet: Ionomycin, thapsigargin Calyculin A, and Cytochalasin D were from Calbiochem (La Jolla, CA).

    Techniques: Staining, Incubation, Fluorescence

    Signaling events, the F-actin cytoskeleton, and lipid rafts are involved in F1845-induced PS externalization on PLB-985 cells. Before challenge with the different E. coli strains, PLB-985 cells were pretreated for 1 h with several modulating agents, including the tyrosine kinase inhibitor genistein, the lipid raft-modifying drug MβCD, the actin cytoskeleton inhibitor cytochalasin D, and the protein kinase C inhibitor staurosporine. At the end of the incubation period the cells were stained with both annexin V-PE and 7-AAD, and the percentages of 7-AAD-negative, annexin V-positive cells were determined by flow cytometry. The data are data from five experiments and are means ± standard errors of the means. *, P

    Journal: Infection and Immunity

    Article Title: Afa/Dr-Expressing, Diffusely Adhering Escherichia coli Strain C1845 Triggers F1845 Fimbria-Dependent Phosphatidylserine Externalization on Neutrophil-Like Differentiated PLB-985 Cells through an Apoptosis-Independent Mechanism ▿

    doi: 10.1128/IAI.01354-09

    Figure Lengend Snippet: Signaling events, the F-actin cytoskeleton, and lipid rafts are involved in F1845-induced PS externalization on PLB-985 cells. Before challenge with the different E. coli strains, PLB-985 cells were pretreated for 1 h with several modulating agents, including the tyrosine kinase inhibitor genistein, the lipid raft-modifying drug MβCD, the actin cytoskeleton inhibitor cytochalasin D, and the protein kinase C inhibitor staurosporine. At the end of the incubation period the cells were stained with both annexin V-PE and 7-AAD, and the percentages of 7-AAD-negative, annexin V-positive cells were determined by flow cytometry. The data are data from five experiments and are means ± standard errors of the means. *, P

    Article Snippet: The following signaling inhibitors were obtained from Sigma: the protein kinase C (PKC) inhibitor staurosporine, the tyrosine kinase inhibitor genistein, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294,002, the lipid raft-modifying drug methyl-β-cyclodextrin (MβCD), and the actin cytoskeleton inhibitor cytochalasin D. The Src family kinase inhibitor PP2 and the MEK/ERK inhibitor PD 98059 were obtained from Calbiochem, and the p38 MAPK inhibitor SB-203580 was obtained from Cell Signaling Technology (Beverly, MA).

    Techniques: Incubation, Staining, Flow Cytometry, Cytometry

    Effects of actin manipulation on oocyte effective tension. (A) Phalloidin staining of a GVI oocyte (left) and a metaphase II (MII) egg (right). Polarity develops during meiotic maturation, such that the metaphase II egg has an actin-rich cap over the meiotic spindle. Red, actin; blue, DNA. Scale bar, (left) 20 μm; (right) 22 μm. (B) Effects on effective tension (T eff ) of actin manipulation by cytochalasin D treatment of GVI oocytes (*p

    Journal: Molecular Biology of the Cell

    Article Title: Cortical Mechanics and Meiosis II Completion in Mammalian Oocytes Are Mediated by Myosin-II and Ezrin-Radixin-Moesin (ERM) Proteins

    doi: 10.1091/mbc.E10-01-0066

    Figure Lengend Snippet: Effects of actin manipulation on oocyte effective tension. (A) Phalloidin staining of a GVI oocyte (left) and a metaphase II (MII) egg (right). Polarity develops during meiotic maturation, such that the metaphase II egg has an actin-rich cap over the meiotic spindle. Red, actin; blue, DNA. Scale bar, (left) 20 μm; (right) 22 μm. (B) Effects on effective tension (T eff ) of actin manipulation by cytochalasin D treatment of GVI oocytes (*p

    Article Snippet: Treatment with the actin filament disruptor cytochalasin D (5 μg/ml; Calbiochem, Gibbstown, NJ) or the myosin light-chain kinase inhibitor ML-7 (15 μM; Calbiochem, La Jolla, CA; or Sigma-Aldrich) was done as previously described ( ; ) for 60 min before and during Teff measurement by MPA.

    Techniques: Staining

    Spindle defects upon exit from metaphase II arrest with actin, myosin-II, or ERM disruption. Eggs were inseminated for 1.5 h (A–P) or 4 h (Q-BB). Control fertilized eggs (A–C, J–M, and Q–T) show normal spindle rotation and second polar body morphology, as illustrated in the schematic diagram. A failure in spindle rotation was observed in eggs treated with the MLCK inhibitor ML-7 (D–F; 73/75 eggs) and in eggs treated with the actin filament disruptor cytochalasin D (G–I; 34/34 eggs). Distorted, curved spindles were observed in DN-RDX–expressing eggs (N–P, U-BB; 26/36 eggs). In these embryos, two polar body-like (PBL) structures formed; these PBL structures did not resolve with increased time after insemination (U-BB). Arrowheads identify polar bodies (PB) and PBL structures. Maternal DNA is labeled m, and sperm DNA is labeled s; some eggs are polyspermic, although this is not uncommon with the insemination conditions used, particularly with cytochalasin D–treated eggs ( McAvey et al. , 2002 ). In several panels, DNA of the fertilizing sperm is out of the plane of focus. FC (T) identifies the fertilization cone containing the DNA of a fertilizing sperm. Scale bar, (DD) 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Cortical Mechanics and Meiosis II Completion in Mammalian Oocytes Are Mediated by Myosin-II and Ezrin-Radixin-Moesin (ERM) Proteins

    doi: 10.1091/mbc.E10-01-0066

    Figure Lengend Snippet: Spindle defects upon exit from metaphase II arrest with actin, myosin-II, or ERM disruption. Eggs were inseminated for 1.5 h (A–P) or 4 h (Q-BB). Control fertilized eggs (A–C, J–M, and Q–T) show normal spindle rotation and second polar body morphology, as illustrated in the schematic diagram. A failure in spindle rotation was observed in eggs treated with the MLCK inhibitor ML-7 (D–F; 73/75 eggs) and in eggs treated with the actin filament disruptor cytochalasin D (G–I; 34/34 eggs). Distorted, curved spindles were observed in DN-RDX–expressing eggs (N–P, U-BB; 26/36 eggs). In these embryos, two polar body-like (PBL) structures formed; these PBL structures did not resolve with increased time after insemination (U-BB). Arrowheads identify polar bodies (PB) and PBL structures. Maternal DNA is labeled m, and sperm DNA is labeled s; some eggs are polyspermic, although this is not uncommon with the insemination conditions used, particularly with cytochalasin D–treated eggs ( McAvey et al. , 2002 ). In several panels, DNA of the fertilizing sperm is out of the plane of focus. FC (T) identifies the fertilization cone containing the DNA of a fertilizing sperm. Scale bar, (DD) 10 μm.

    Article Snippet: Treatment with the actin filament disruptor cytochalasin D (5 μg/ml; Calbiochem, Gibbstown, NJ) or the myosin light-chain kinase inhibitor ML-7 (15 μM; Calbiochem, La Jolla, CA; or Sigma-Aldrich) was done as previously described ( ; ) for 60 min before and during Teff measurement by MPA.

    Techniques: Expressing, Labeling