cell lysis buffer  (Bio-Rad)

 
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    Name:
    Lysis Buffer
    Description:
    100 ml refill reagent for use with Genes in a Bottle Kit 166 2300EDU education use only
    Catalog Number:
    1662002EDU
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    None
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    Refresh Kit Components
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    Structured Review

    Bio-Rad cell lysis buffer
    Lysis Buffer
    100 ml refill reagent for use with Genes in a Bottle Kit 166 2300EDU education use only
    https://www.bioz.com/result/cell lysis buffer/product/Bio-Rad
    Average 99 stars, based on 176 article reviews
    Price from $9.99 to $1999.99
    cell lysis buffer - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins
    Article Snippet: .. The nuclei/membrane fraction were dissolved in 2-DE lysis buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 1% DTT, and 0.8% pharmalyte and protease inhibitor (all from Bio-Rad) on ice for 30 min. After centrifugation at 15000 ×g for 30 min at 4°C, the supernatant containing solubilized nuclei/membrane proteins could be used for 2-DE analysis. ..

    Article Title: Discovery of Novel Small-Molecule Compounds with Selective Cytotoxicity for Burkitt’s Lymphoma Cells Using 3D Ligand-Based Virtual Screening
    Article Snippet: .. The Ramos cells incubated with vehicle or compounds for the indicated times were washed twice in PBS and resuspended in 200 µL ice-cold caspase lysis buffer (0.1% Triton X-100, 100 mM phosphate buffer, pH 6.0, 1.3 mM EDTA, 100 mM NaCl), sonicated, and left on ice for 30 min. After centrifugation (14,000× g , 15 min, 4 °C), the total protein concentrations in the supernatants were measured using BioRad Protein Assay kits (Bio-Rad, Hercules, CA, USA), according to the manufacturer instructions. .. Cell lysates (20 μg protein) were incubated for 30 min at 37 °C in caspase reaction buffer (20 mM PIPES, pH 7.2, 10% sucrose, 0.1% CHAPS, 1 mM EDTA, 100 mM NaCl), after which 100 µM Ac-DEVD-AFC peptide substrate was added.

    Protease Inhibitor:

    Article Title: Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins
    Article Snippet: .. The nuclei/membrane fraction were dissolved in 2-DE lysis buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 1% DTT, and 0.8% pharmalyte and protease inhibitor (all from Bio-Rad) on ice for 30 min. After centrifugation at 15000 ×g for 30 min at 4°C, the supernatant containing solubilized nuclei/membrane proteins could be used for 2-DE analysis. ..

    Protein Concentration:

    Article Title: AdipoRon, the first orally active adiponectin receptor activator, attenuates postischemic myocardial apoptosis through both AMPK-mediated and AMPK-independent signalings
    Article Snippet: .. Briefly, cells or mouse heart tissue were lysed with 1× caspase-3 lysis buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 5 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100), and total protein concentration was determined by the Bradford method (Bio-Rad). .. To each well of a 96-well plate, supernatant containing 50 μg/50 μl protein was loaded and incubated with 3.645 μg of Ac-DEVD-AFC (Biomol, P-409) in 50 μl of 2× assay buffer (100 mM HEPES, 200 mM NaCl, 0.2% CHAPS, 2 mM EDTA,10% glycerol, 10 mM DTT, pH7.4) at 37°C for 1.5 h. AFC was cleaved from Ac-DEVD-AFC by activated caspase-3, and the free AFC was quantified with a Spectra Max M5 fluorescence microplate reader (excitation wavelength, 400 nm, emission wavelength, 508 nm; Molecular Devices, Sunnyvale, CA) by AFC standard curve (Biomol, KI-108).

    Infection:

    Article Title: Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3−/− Macrophages
    Article Snippet: .. Probing the RT Profiler Human Inflammasome PCR Arrays THP-1 cells, with and without T. cruzi infection and treatments, were harvested with 500 µl Bio-Rad cell lysis/RNA extraction buffer. .. Total RNA was extracted by using an Aurum DNA-free RNA isolation kit (Bio-Rad, Hercules, CA) and measured at 260 and 280 nm for determination of purity and concentration.

    Incubation:

    Article Title: Discovery of Novel Small-Molecule Compounds with Selective Cytotoxicity for Burkitt’s Lymphoma Cells Using 3D Ligand-Based Virtual Screening
    Article Snippet: .. The Ramos cells incubated with vehicle or compounds for the indicated times were washed twice in PBS and resuspended in 200 µL ice-cold caspase lysis buffer (0.1% Triton X-100, 100 mM phosphate buffer, pH 6.0, 1.3 mM EDTA, 100 mM NaCl), sonicated, and left on ice for 30 min. After centrifugation (14,000× g , 15 min, 4 °C), the total protein concentrations in the supernatants were measured using BioRad Protein Assay kits (Bio-Rad, Hercules, CA, USA), according to the manufacturer instructions. .. Cell lysates (20 μg protein) were incubated for 30 min at 37 °C in caspase reaction buffer (20 mM PIPES, pH 7.2, 10% sucrose, 0.1% CHAPS, 1 mM EDTA, 100 mM NaCl), after which 100 µM Ac-DEVD-AFC peptide substrate was added.

    Bradford Protein Assay:

    Article Title: Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay
    Article Snippet: .. Extracts of total protein for analysis by 2D-DiGE were obtained from tissue culture cells using ASB14 lysis buffer and their concentrations quantified by DC Bradford protein assay (Biorad). .. Protein separation was performed using a pI range of pH3-10 (non-linear strip) and a 12.5% SDS-PAGE gel.

    Western Blot:

    Article Title: PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation
    Article Snippet: .. Western blotting HepaRG cells were washed twice in PBS, lysed in 2× SDS lysis buffer (150 mM Tris-HCl, pH 6.8, 25% glycerol, 5% SDS, 0.01% Bromophenol Blue), sonicated briefly to shear DNA and protein estimation performed using the DC protein estimation kit (Bio-Rad). .. NB4 cells were lysed in 8 M Urea with 0.1 M dithiothreitol, sonicated briefly and protein concentration estimated using the Bio-Rad protein assay.

    Polymerase Chain Reaction:

    Article Title: Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3−/− Macrophages
    Article Snippet: .. Probing the RT Profiler Human Inflammasome PCR Arrays THP-1 cells, with and without T. cruzi infection and treatments, were harvested with 500 µl Bio-Rad cell lysis/RNA extraction buffer. .. Total RNA was extracted by using an Aurum DNA-free RNA isolation kit (Bio-Rad, Hercules, CA) and measured at 260 and 280 nm for determination of purity and concentration.

    Sonication:

    Article Title: Discovery of Novel Small-Molecule Compounds with Selective Cytotoxicity for Burkitt’s Lymphoma Cells Using 3D Ligand-Based Virtual Screening
    Article Snippet: .. The Ramos cells incubated with vehicle or compounds for the indicated times were washed twice in PBS and resuspended in 200 µL ice-cold caspase lysis buffer (0.1% Triton X-100, 100 mM phosphate buffer, pH 6.0, 1.3 mM EDTA, 100 mM NaCl), sonicated, and left on ice for 30 min. After centrifugation (14,000× g , 15 min, 4 °C), the total protein concentrations in the supernatants were measured using BioRad Protein Assay kits (Bio-Rad, Hercules, CA, USA), according to the manufacturer instructions. .. Cell lysates (20 μg protein) were incubated for 30 min at 37 °C in caspase reaction buffer (20 mM PIPES, pH 7.2, 10% sucrose, 0.1% CHAPS, 1 mM EDTA, 100 mM NaCl), after which 100 µM Ac-DEVD-AFC peptide substrate was added.

    Article Title: PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation
    Article Snippet: .. Western blotting HepaRG cells were washed twice in PBS, lysed in 2× SDS lysis buffer (150 mM Tris-HCl, pH 6.8, 25% glycerol, 5% SDS, 0.01% Bromophenol Blue), sonicated briefly to shear DNA and protein estimation performed using the DC protein estimation kit (Bio-Rad). .. NB4 cells were lysed in 8 M Urea with 0.1 M dithiothreitol, sonicated briefly and protein concentration estimated using the Bio-Rad protein assay.

    Lysis:

    Article Title: Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins
    Article Snippet: .. The nuclei/membrane fraction were dissolved in 2-DE lysis buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 1% DTT, and 0.8% pharmalyte and protease inhibitor (all from Bio-Rad) on ice for 30 min. After centrifugation at 15000 ×g for 30 min at 4°C, the supernatant containing solubilized nuclei/membrane proteins could be used for 2-DE analysis. ..

    Article Title: AdipoRon, the first orally active adiponectin receptor activator, attenuates postischemic myocardial apoptosis through both AMPK-mediated and AMPK-independent signalings
    Article Snippet: .. Briefly, cells or mouse heart tissue were lysed with 1× caspase-3 lysis buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 5 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100), and total protein concentration was determined by the Bradford method (Bio-Rad). .. To each well of a 96-well plate, supernatant containing 50 μg/50 μl protein was loaded and incubated with 3.645 μg of Ac-DEVD-AFC (Biomol, P-409) in 50 μl of 2× assay buffer (100 mM HEPES, 200 mM NaCl, 0.2% CHAPS, 2 mM EDTA,10% glycerol, 10 mM DTT, pH7.4) at 37°C for 1.5 h. AFC was cleaved from Ac-DEVD-AFC by activated caspase-3, and the free AFC was quantified with a Spectra Max M5 fluorescence microplate reader (excitation wavelength, 400 nm, emission wavelength, 508 nm; Molecular Devices, Sunnyvale, CA) by AFC standard curve (Biomol, KI-108).

    Article Title: Discovery of Novel Small-Molecule Compounds with Selective Cytotoxicity for Burkitt’s Lymphoma Cells Using 3D Ligand-Based Virtual Screening
    Article Snippet: .. The Ramos cells incubated with vehicle or compounds for the indicated times were washed twice in PBS and resuspended in 200 µL ice-cold caspase lysis buffer (0.1% Triton X-100, 100 mM phosphate buffer, pH 6.0, 1.3 mM EDTA, 100 mM NaCl), sonicated, and left on ice for 30 min. After centrifugation (14,000× g , 15 min, 4 °C), the total protein concentrations in the supernatants were measured using BioRad Protein Assay kits (Bio-Rad, Hercules, CA, USA), according to the manufacturer instructions. .. Cell lysates (20 μg protein) were incubated for 30 min at 37 °C in caspase reaction buffer (20 mM PIPES, pH 7.2, 10% sucrose, 0.1% CHAPS, 1 mM EDTA, 100 mM NaCl), after which 100 µM Ac-DEVD-AFC peptide substrate was added.

    Article Title: A single lysis solution for the analysis of tissue samples by different proteomic technologies), A single lysis solution for the analysis of tissue samples by different proteomic technologies
    Article Snippet: .. Typically, the IPG strips were actively rehydrated in 300μl of sample solution, CLB1 or IPG lysis buffer (Bio‐Rad Laboratories, USA). .. IEF was then carried out for 18,000Vhr.

    Article Title: PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation
    Article Snippet: .. Western blotting HepaRG cells were washed twice in PBS, lysed in 2× SDS lysis buffer (150 mM Tris-HCl, pH 6.8, 25% glycerol, 5% SDS, 0.01% Bromophenol Blue), sonicated briefly to shear DNA and protein estimation performed using the DC protein estimation kit (Bio-Rad). .. NB4 cells were lysed in 8 M Urea with 0.1 M dithiothreitol, sonicated briefly and protein concentration estimated using the Bio-Rad protein assay.

    Article Title: Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay
    Article Snippet: .. Extracts of total protein for analysis by 2D-DiGE were obtained from tissue culture cells using ASB14 lysis buffer and their concentrations quantified by DC Bradford protein assay (Biorad). .. Protein separation was performed using a pI range of pH3-10 (non-linear strip) and a 12.5% SDS-PAGE gel.

    Article Title: Analysis of Cytoplasmic and Secreted Proteins of Staphylococcus aureus Revealed Adaptive Metabolic Homeostasis in Response to Changes in the Environmental Conditions Representative of the Human Wound Site
    Article Snippet: .. The samples were air-dried, and each replicate was resolved in a 500 μL SDS-PAGE loading buffer SDS Lysis Buffer containing protein concentrations in each sample, which were determined using the BCATM assay (Bio-Rad) following the manufacturer’s instructions, and bovine serum albumin (BSA) was used as the reference standard. .. Fifteen micrograms of protein from each sample was run in SDS-PAGE to determine any large-scale differences in the protein production between the control and treated cells.

    Article Title: Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3−/− Macrophages
    Article Snippet: .. Probing the RT Profiler Human Inflammasome PCR Arrays THP-1 cells, with and without T. cruzi infection and treatments, were harvested with 500 µl Bio-Rad cell lysis/RNA extraction buffer. .. Total RNA was extracted by using an Aurum DNA-free RNA isolation kit (Bio-Rad, Hercules, CA) and measured at 260 and 280 nm for determination of purity and concentration.

    SDS Page:

    Article Title: Analysis of Cytoplasmic and Secreted Proteins of Staphylococcus aureus Revealed Adaptive Metabolic Homeostasis in Response to Changes in the Environmental Conditions Representative of the Human Wound Site
    Article Snippet: .. The samples were air-dried, and each replicate was resolved in a 500 μL SDS-PAGE loading buffer SDS Lysis Buffer containing protein concentrations in each sample, which were determined using the BCATM assay (Bio-Rad) following the manufacturer’s instructions, and bovine serum albumin (BSA) was used as the reference standard. .. Fifteen micrograms of protein from each sample was run in SDS-PAGE to determine any large-scale differences in the protein production between the control and treated cells.

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  • 94
    Bio-Rad il 1β
    Flow cytometric analysis of cytokine-stimulated CD62E (E-selectin), CD54 (ICAM-1) and CD106 (VCAM-1) expression by HUVECs. HUVECs was cultivated with <t>IL-1β</t> (1 ng/mL), TNF-β (10 ng/mL), TNF-α (10 ng/mL), IL-6 (10 ng/mL), IFN-γ (10 ng/mL), ECGS (100 μg/mL) and fibronectin (100 ng/mL), respectively, for 4 h. Expression of CD62E, CD54 and CD106 were detected by flow cytometry using silver area as isotype controls. Bar graph shows the percentage of positive cells (right panels), contour polt analysis (left panels) shows CD62E + , CD54 + and CD106 + cells, in comparison with isotype controls. Bars represent the mean ± SEM (n = 3).
    Il 1β, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad tris glycine polyacrylamide gels
    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% <t>Bis-Tris</t> gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL <t>PVDF</t> membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin ( Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies ( Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
    Tris Glycine Polyacrylamide Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad triton x 100 buffer
    TSPAN12(Pal − ) mutant associates with wild-type TSPAN12 and perturbs its subcellular distribution.  A ) TSPAN12(Pal − )-GFP (lanes 2–5, 7–10) or CD81(Pal − )-GFP (lanes 6, 11) were expressed in MCF7 cells, together with vector-FLAG (lanes 5, 10) or other FLAG-tagged proteins (lanes 2–4, 6, 8, 9, 11). Lanes 1 and 7 are from untransfected MCF7 (no GFP or FLAG proteins). After lysis (1% Triton X-100) MCF7 cells were treated with (lanes 7–11) or without (lanes 1–6) covalent cross-linker DSP. After anti-FLAG immunoprecipitations, reduction of the dithiol cross-link, and SDS-PAGE, proteins were detected by GFP immunoblotting. Diffuse proteins of 50–60 kDa in lanes 5, 9, and 10 likely represent background proteins, immunoprecipitated by anti-FLAG antibody (even when no FLAG proteins are present, as in lanes 5, 10), which also weakly cross-react with anti-GFP antibody.  B ) Either TSPAN12-GFP ( a – c ) or TSPAN12(Pal − )-GFP ( d ) was expressed in MCF7 cells, together with indicated FLAG-tagged proteins. FLAG- and GFP-tagged tetraspanins were expressed at comparable levels, as seen by immunoblotting (not shown).
    Triton X 100 Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad cell lysis rna extraction buffer
    Venn diagram of inflammasome-related differential gene expression in mφs infected with T. <t>cruzi</t> (±ATP). THP-1 mφs were incubated with T. cruzi or LPS (± ATP treatment) as in Fig. 1 . Total <t>RNA</t> was isolated, and cDNA was used as a template to probe the expression of 95 genes (including house-keeping genes) in the RT 2 Profiler Inflammasome PCR Arrays. The differential mRNA level was captured by quantitative RT-PCR, normalized to housekeeping genes, and HTqPCR was employed to attain the statistically significant differential expression in treated- versus-control samples ( Table 1 and Table S2 ). Shown are Venn diagrams of comparative analysis of gene expression in T. cruzi -infected mφs at 3 h versus 18 h (A) , effect of ATP stimulus on gene expression at 3 h (B) and 18 h (C) pi, and comparative effect of ATP stimulus on gene expression in LPS-treated mφs at 3 h (D) and 18 h (E) . Differential up-regulation (green) and down-regulation (red) of genes with respect to controls is presented. Genes presenting as red with green arrow in B – F showed decreased expression without ATP but were up-regulated by ATP treatment (and vice versa).
    Cell Lysis Rna Extraction Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Flow cytometric analysis of cytokine-stimulated CD62E (E-selectin), CD54 (ICAM-1) and CD106 (VCAM-1) expression by HUVECs. HUVECs was cultivated with IL-1β (1 ng/mL), TNF-β (10 ng/mL), TNF-α (10 ng/mL), IL-6 (10 ng/mL), IFN-γ (10 ng/mL), ECGS (100 μg/mL) and fibronectin (100 ng/mL), respectively, for 4 h. Expression of CD62E, CD54 and CD106 were detected by flow cytometry using silver area as isotype controls. Bar graph shows the percentage of positive cells (right panels), contour polt analysis (left panels) shows CD62E + , CD54 + and CD106 + cells, in comparison with isotype controls. Bars represent the mean ± SEM (n = 3).

    Journal: Scientific Reports

    Article Title: Nitric Oxide Inhibits Hetero-adhesion of Cancer Cells to Endothelial Cells: Restraining Circulating Tumor Cells from Initiating Metastatic Cascade

    doi: 10.1038/srep04344

    Figure Lengend Snippet: Flow cytometric analysis of cytokine-stimulated CD62E (E-selectin), CD54 (ICAM-1) and CD106 (VCAM-1) expression by HUVECs. HUVECs was cultivated with IL-1β (1 ng/mL), TNF-β (10 ng/mL), TNF-α (10 ng/mL), IL-6 (10 ng/mL), IFN-γ (10 ng/mL), ECGS (100 μg/mL) and fibronectin (100 ng/mL), respectively, for 4 h. Expression of CD62E, CD54 and CD106 were detected by flow cytometry using silver area as isotype controls. Bar graph shows the percentage of positive cells (right panels), contour polt analysis (left panels) shows CD62E + , CD54 + and CD106 + cells, in comparison with isotype controls. Bars represent the mean ± SEM (n = 3).

    Article Snippet: Western blot analysis To induce the expression of VCAM-1, HUVECs were incubated with fresh media (without ECGS), and pretreated with IL-1β (1 ng/mL) or IFN-γ (10 ng/mL) for 4 h, followed by treatment of the HUVECs with CAP-NO for 12 h. After cell lysis, equal volumes (20 μL per lane) of protein were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad), and transferred to polyvinylidene difluoride (PVDF, Bio-Rad) membrane.

    Techniques: Flow Cytometry, Expressing, Cytometry

    CAP-NO inhibited adhesion of HT-29 cells to HUVECs. (a) Effect of CAP-NO on the spontaneous adhesion of HT-29 cells to culture plate within 1 h observation. (b) Quantification of HT-29 adhered to the HUVEC monolayers in the presence and absence of CAP-NO or CAP. (c) Effects of CAP-NO on adhesion of HT-29 to HUVECs at indicated time points. The % relative adhesion was determined by fluorescence-labeled cell count assay, and the results are based on the IL-1β stimulated HUVECs. (d) Rhodamine 123-labeled HT-29 cells were added to the HUVEC monolayers stimulated by IL-1β (1 ng/mL) in the presence and absence of CAP-NO or CAP (both 50 μM). Fluorescence microscopy showed HT-29 cells (green) adhered to the HUVECs. Bars represent the mean ± SEM (n = 3); ** indicates P

    Journal: Scientific Reports

    Article Title: Nitric Oxide Inhibits Hetero-adhesion of Cancer Cells to Endothelial Cells: Restraining Circulating Tumor Cells from Initiating Metastatic Cascade

    doi: 10.1038/srep04344

    Figure Lengend Snippet: CAP-NO inhibited adhesion of HT-29 cells to HUVECs. (a) Effect of CAP-NO on the spontaneous adhesion of HT-29 cells to culture plate within 1 h observation. (b) Quantification of HT-29 adhered to the HUVEC monolayers in the presence and absence of CAP-NO or CAP. (c) Effects of CAP-NO on adhesion of HT-29 to HUVECs at indicated time points. The % relative adhesion was determined by fluorescence-labeled cell count assay, and the results are based on the IL-1β stimulated HUVECs. (d) Rhodamine 123-labeled HT-29 cells were added to the HUVEC monolayers stimulated by IL-1β (1 ng/mL) in the presence and absence of CAP-NO or CAP (both 50 μM). Fluorescence microscopy showed HT-29 cells (green) adhered to the HUVECs. Bars represent the mean ± SEM (n = 3); ** indicates P

    Article Snippet: Western blot analysis To induce the expression of VCAM-1, HUVECs were incubated with fresh media (without ECGS), and pretreated with IL-1β (1 ng/mL) or IFN-γ (10 ng/mL) for 4 h, followed by treatment of the HUVECs with CAP-NO for 12 h. After cell lysis, equal volumes (20 μL per lane) of protein were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad), and transferred to polyvinylidene difluoride (PVDF, Bio-Rad) membrane.

    Techniques: Fluorescence, Labeling, Cell Counting, Microscopy

    Flow cytometric and western blot analyses of effects of CAP-NO and CAP on CAMs. HUVECs were pretreated with 1 ng/mL IL-1β for 4 h followed by incubation with CAP-NO or CAP for another 12 h (unless otherwise stated). Flow cytometric analysis showed significant inhibition by CAP-NO on expression induced by IL-1β (1 ng/mL) of ICAM-1 (a), E-selectin (b), and VCAM-1 (c). Results are expressed as the mean fluorescence intensities and calculated as percentage of controls. (d) Effects of CAP-NO on E-selectin expression at indicated time points. (e) Expression of VCAM-1 determined by flow cytometry using green curve as a control (IL-1β only), and silver area is an isotype control. (f) Effects of CAP-NO on CD29 expression at indicated time points. (g and h) Western blot analysis of VCAM-1 expression by HUVECs pretreated with IFN-γ (10 ng/mL) or IL-1β (1 ng/mL) for 4 h followed by incubation with CAP-NO (0, 1, 10 and 100 μM) for another 12 h. Band intensity was quantified by using Image Lab analysis software, and calculated as percentage of controls (IL-1β only). Bars represent the mean ± SEM (n = 3); *, P

    Journal: Scientific Reports

    Article Title: Nitric Oxide Inhibits Hetero-adhesion of Cancer Cells to Endothelial Cells: Restraining Circulating Tumor Cells from Initiating Metastatic Cascade

    doi: 10.1038/srep04344

    Figure Lengend Snippet: Flow cytometric and western blot analyses of effects of CAP-NO and CAP on CAMs. HUVECs were pretreated with 1 ng/mL IL-1β for 4 h followed by incubation with CAP-NO or CAP for another 12 h (unless otherwise stated). Flow cytometric analysis showed significant inhibition by CAP-NO on expression induced by IL-1β (1 ng/mL) of ICAM-1 (a), E-selectin (b), and VCAM-1 (c). Results are expressed as the mean fluorescence intensities and calculated as percentage of controls. (d) Effects of CAP-NO on E-selectin expression at indicated time points. (e) Expression of VCAM-1 determined by flow cytometry using green curve as a control (IL-1β only), and silver area is an isotype control. (f) Effects of CAP-NO on CD29 expression at indicated time points. (g and h) Western blot analysis of VCAM-1 expression by HUVECs pretreated with IFN-γ (10 ng/mL) or IL-1β (1 ng/mL) for 4 h followed by incubation with CAP-NO (0, 1, 10 and 100 μM) for another 12 h. Band intensity was quantified by using Image Lab analysis software, and calculated as percentage of controls (IL-1β only). Bars represent the mean ± SEM (n = 3); *, P

    Article Snippet: Western blot analysis To induce the expression of VCAM-1, HUVECs were incubated with fresh media (without ECGS), and pretreated with IL-1β (1 ng/mL) or IFN-γ (10 ng/mL) for 4 h, followed by treatment of the HUVECs with CAP-NO for 12 h. After cell lysis, equal volumes (20 μL per lane) of protein were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad), and transferred to polyvinylidene difluoride (PVDF, Bio-Rad) membrane.

    Techniques: Flow Cytometry, Western Blot, Incubation, Inhibition, Expressing, Fluorescence, Cytometry, Software

    NO scavenger Hb reversed the inhibitory effect of CAP-NO on the hetero-adhesion between HT-29 and HUVECs and -induced expression of VCAM-1 in the presence of IL-1β (1 ng/mL). (a) 5-min pretreatment with Hb (1 μM) reversed the inhibition CAP-NO on the IL-1β-induced adhesion determined by fluorescence-labeled cell count assay. (b) Hb also reversed IL-1β-induced expression of VCAM-1 (CD106). Results are expressed as the mean fluorescence intensities and calculated as percentage of controls (IL-1β only). Bars represent the mean ± SEM (n = 3).

    Journal: Scientific Reports

    Article Title: Nitric Oxide Inhibits Hetero-adhesion of Cancer Cells to Endothelial Cells: Restraining Circulating Tumor Cells from Initiating Metastatic Cascade

    doi: 10.1038/srep04344

    Figure Lengend Snippet: NO scavenger Hb reversed the inhibitory effect of CAP-NO on the hetero-adhesion between HT-29 and HUVECs and -induced expression of VCAM-1 in the presence of IL-1β (1 ng/mL). (a) 5-min pretreatment with Hb (1 μM) reversed the inhibition CAP-NO on the IL-1β-induced adhesion determined by fluorescence-labeled cell count assay. (b) Hb also reversed IL-1β-induced expression of VCAM-1 (CD106). Results are expressed as the mean fluorescence intensities and calculated as percentage of controls (IL-1β only). Bars represent the mean ± SEM (n = 3).

    Article Snippet: Western blot analysis To induce the expression of VCAM-1, HUVECs were incubated with fresh media (without ECGS), and pretreated with IL-1β (1 ng/mL) or IFN-γ (10 ng/mL) for 4 h, followed by treatment of the HUVECs with CAP-NO for 12 h. After cell lysis, equal volumes (20 μL per lane) of protein were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad), and transferred to polyvinylidene difluoride (PVDF, Bio-Rad) membrane.

    Techniques: Expressing, Inhibition, Fluorescence, Labeling, Cell Counting

    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin ( Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies ( Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).

    Journal: eLife

    Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

    doi: 10.7554/eLife.40033

    Figure Lengend Snippet: Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin ( Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies ( Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).

    Article Snippet: Proteins were fractionated on 4–15% Tris-glycine polyacrylamide gels (Criterion gels, Bio-Rad), transferred to PVDF membranes, and probed with antibodies: FGFR1, fibronectin (Cell Signaling, Danvers, MA, USA); CD9, FGF2, calreticulin, tsg101 (Santa Cruz Biotechnology, Dallas, TX, USA), CD63 (Abcam, Boston, MA, USA), and actin (MAB1501, Millipore, Burlington, MA, USA).

    Techniques: Cell Culture, Lysis, Protease Inhibitor, Centrifugation, Incubation, Imaging

    TSPAN12(Pal − ) mutant associates with wild-type TSPAN12 and perturbs its subcellular distribution.  A ) TSPAN12(Pal − )-GFP (lanes 2–5, 7–10) or CD81(Pal − )-GFP (lanes 6, 11) were expressed in MCF7 cells, together with vector-FLAG (lanes 5, 10) or other FLAG-tagged proteins (lanes 2–4, 6, 8, 9, 11). Lanes 1 and 7 are from untransfected MCF7 (no GFP or FLAG proteins). After lysis (1% Triton X-100) MCF7 cells were treated with (lanes 7–11) or without (lanes 1–6) covalent cross-linker DSP. After anti-FLAG immunoprecipitations, reduction of the dithiol cross-link, and SDS-PAGE, proteins were detected by GFP immunoblotting. Diffuse proteins of 50–60 kDa in lanes 5, 9, and 10 likely represent background proteins, immunoprecipitated by anti-FLAG antibody (even when no FLAG proteins are present, as in lanes 5, 10), which also weakly cross-react with anti-GFP antibody.  B ) Either TSPAN12-GFP ( a – c ) or TSPAN12(Pal − )-GFP ( d ) was expressed in MCF7 cells, together with indicated FLAG-tagged proteins. FLAG- and GFP-tagged tetraspanins were expressed at comparable levels, as seen by immunoblotting (not shown).

    Journal: The FASEB Journal

    Article Title: Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein

    doi: 10.1096/fj.09-133462

    Figure Lengend Snippet: TSPAN12(Pal − ) mutant associates with wild-type TSPAN12 and perturbs its subcellular distribution. A ) TSPAN12(Pal − )-GFP (lanes 2–5, 7–10) or CD81(Pal − )-GFP (lanes 6, 11) were expressed in MCF7 cells, together with vector-FLAG (lanes 5, 10) or other FLAG-tagged proteins (lanes 2–4, 6, 8, 9, 11). Lanes 1 and 7 are from untransfected MCF7 (no GFP or FLAG proteins). After lysis (1% Triton X-100) MCF7 cells were treated with (lanes 7–11) or without (lanes 1–6) covalent cross-linker DSP. After anti-FLAG immunoprecipitations, reduction of the dithiol cross-link, and SDS-PAGE, proteins were detected by GFP immunoblotting. Diffuse proteins of 50–60 kDa in lanes 5, 9, and 10 likely represent background proteins, immunoprecipitated by anti-FLAG antibody (even when no FLAG proteins are present, as in lanes 5, 10), which also weakly cross-react with anti-GFP antibody. B ) Either TSPAN12-GFP ( a – c ) or TSPAN12(Pal − )-GFP ( d ) was expressed in MCF7 cells, together with indicated FLAG-tagged proteins. FLAG- and GFP-tagged tetraspanins were expressed at comparable levels, as seen by immunoblotting (not shown).

    Article Snippet: To confirm cell equivalency, cells were lysed (1% Triton X-100 buffer), and proteins were quantified using a DC Protein Assay kit (Bio-Rad, Hercules, CA, USA).

    Techniques: Mutagenesis, Plasmid Preparation, Lysis, SDS Page, Immunoprecipitation

    Venn diagram of inflammasome-related differential gene expression in mφs infected with T. cruzi (±ATP). THP-1 mφs were incubated with T. cruzi or LPS (± ATP treatment) as in Fig. 1 . Total RNA was isolated, and cDNA was used as a template to probe the expression of 95 genes (including house-keeping genes) in the RT 2 Profiler Inflammasome PCR Arrays. The differential mRNA level was captured by quantitative RT-PCR, normalized to housekeeping genes, and HTqPCR was employed to attain the statistically significant differential expression in treated- versus-control samples ( Table 1 and Table S2 ). Shown are Venn diagrams of comparative analysis of gene expression in T. cruzi -infected mφs at 3 h versus 18 h (A) , effect of ATP stimulus on gene expression at 3 h (B) and 18 h (C) pi, and comparative effect of ATP stimulus on gene expression in LPS-treated mφs at 3 h (D) and 18 h (E) . Differential up-regulation (green) and down-regulation (red) of genes with respect to controls is presented. Genes presenting as red with green arrow in B – F showed decreased expression without ATP but were up-regulated by ATP treatment (and vice versa).

    Journal: PLoS ONE

    Article Title: Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3−/− Macrophages

    doi: 10.1371/journal.pone.0111539

    Figure Lengend Snippet: Venn diagram of inflammasome-related differential gene expression in mφs infected with T. cruzi (±ATP). THP-1 mφs were incubated with T. cruzi or LPS (± ATP treatment) as in Fig. 1 . Total RNA was isolated, and cDNA was used as a template to probe the expression of 95 genes (including house-keeping genes) in the RT 2 Profiler Inflammasome PCR Arrays. The differential mRNA level was captured by quantitative RT-PCR, normalized to housekeeping genes, and HTqPCR was employed to attain the statistically significant differential expression in treated- versus-control samples ( Table 1 and Table S2 ). Shown are Venn diagrams of comparative analysis of gene expression in T. cruzi -infected mφs at 3 h versus 18 h (A) , effect of ATP stimulus on gene expression at 3 h (B) and 18 h (C) pi, and comparative effect of ATP stimulus on gene expression in LPS-treated mφs at 3 h (D) and 18 h (E) . Differential up-regulation (green) and down-regulation (red) of genes with respect to controls is presented. Genes presenting as red with green arrow in B – F showed decreased expression without ATP but were up-regulated by ATP treatment (and vice versa).

    Article Snippet: Probing the RT Profiler Human Inflammasome PCR Arrays THP-1 cells, with and without T. cruzi infection and treatments, were harvested with 500 µl Bio-Rad cell lysis/RNA extraction buffer.

    Techniques: Expressing, Infection, Incubation, Isolation, Polymerase Chain Reaction, Quantitative RT-PCR