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  • 99
    Millipore amicon ultra
    Examination of <t>ssDNA_ODN</t> amounts in the concentrate and flow-through after centrifugation of water or 5 M urea solutions containing 10 μ g ssDNA_ODN using <t>Amicon</t> Ultra 30K centrifugal filters. Samples of 15 μ L were analyzed on a 10% denaturing urea-polyacrylamide gel. Lane 1: 300 ng ssDNA_ODN (positive control), Lane 2: concentrate of water solution, Lane 3: flow-through of water solution, Lane 4: concentrate of 5 M urea solution, and Lane 5: flow-through of 5 M urea solution.
    Amicon Ultra, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore amicon ultra 15 centrifugal filter
    MDA-MB-231 aggressive breast cancer cells process and secrete PDGF-C into culture medium. A and B , the PDGF-C protein levels in various cancer cell culture supernatants were subjected to Western blot analysis. After 48 h of incubation in serum-free medium, supernatants from MDA-MB-231, MCF-7, MeWo, A549, and HT-29 cells were concentrated using a 10-K <t>Amicon</t> <t>Ultra-15</t> centrifugal filter. Each concentration was subjected to Western blot analysis using anti-PDGF-C antibody under reducing ( A ) and non-reducing ( B ) conditions. The molecular mass (kDa) is indicated on the left side. C , real-time RT-PCR quantification of PDGF-C mRNA expression in various cancer cell lines. GAPDH is used for normalization of levels. Individual error bars represent the mean ( bars ) ± S.D. of triplicates. ***, p
    Amicon Ultra 15 Centrifugal Filter, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8088 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore amicon ultra 4 centrifugal filter
    MDA-MB-231 aggressive breast cancer cells process and secrete PDGF-C into culture medium. A and B , the PDGF-C protein levels in various cancer cell culture supernatants were subjected to Western blot analysis. After 48 h of incubation in serum-free medium, supernatants from MDA-MB-231, MCF-7, MeWo, A549, and HT-29 cells were concentrated using a 10-K <t>Amicon</t> <t>Ultra-15</t> centrifugal filter. Each concentration was subjected to Western blot analysis using anti-PDGF-C antibody under reducing ( A ) and non-reducing ( B ) conditions. The molecular mass (kDa) is indicated on the left side. C , real-time RT-PCR quantification of PDGF-C mRNA expression in various cancer cell lines. GAPDH is used for normalization of levels. Individual error bars represent the mean ( bars ) ± S.D. of triplicates. ***, p
    Amicon Ultra 4 Centrifugal Filter, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3286 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore amicon ultra 4 centrifugal filter devices
    The specific blockade of TβRI kinase ablated Smad2 activation and dramatically affected the induction of the EMT markers, vimentin and MMPs by FK506. HK-2 cells were cultured on 6-well plates and treated with vehicle control, 5 µM FK506 with or without 500 nM of a TGFβRI/ALK5 kinase inhibitor V (SD-208). ( A ) Cells were incubated for 48 h with SD-208 concentrations ranging from 0–20μM. LDH and resazurin assays were conducted to assess SD-208 inhibitor toxicity; ( B ) Whole cell protein was extracted at 48 h with RIPA buffer. Equal amounts of protein were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose and indirectly probed for vimentin and fibronectin using a mAb and ECL detection system. Whole Smad2 and GAPDH was used as a loading control. A representative blot is shown from three independent experiments. Densitometry is shown for the replicate blots; ( C ) Cell culture supernatants were harvested, centrifuged to remove cellular debris and concentrated using <t>Amicon</t> <t>Ultra-4</t> Centrifugal Filter Devices (Millipore). Gelatin substrate zymography was used to separate and assess MMP-9 and MMP-2 activity. Clear areas represent areas of gelatinolytic activity. A representative zymogram is shown from three independent experiments. ** and *** indicates different levels of significant compared to time-matched control; * , ** and *** indicates varying levels of significance compared to treatment + inhibitor.
    Amicon Ultra 4 Centrifugal Filter Devices, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 761 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore amicon centrifugal filters
    MS rats show increased insulin binding to plasmatic proteins . (A) Western blot from plasma of control rats (lane 1, 2) and MS rats (3, 4, 5) that were previously separated using different <t>Amicon</t> centrifugal filters and immunoprecipitated with an insulin receptor (alpha-IR) in a native gel, stained with an anti-insulin antibody. Observe that insulin is present in different molecular weight bands, Section “ Materials and Methods .” (B) Rats with metabolic syndrome (MS) show increased insulin levels when compared to control rats (C). (C) MS rats display higher insulin levels in the plasmatic fraction containing proteins above 30 <t>kDa,</t> suggesting higher insulin binding to plasmatic proteins. Bars represent mean ± SEM of control ( n = 12) and MS rats ( n = 7), * p
    Amicon Centrifugal Filters, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1055 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore amicon ultra 0 5 centrifugal filter
    Effects of MAB treatment on dystrophin expression Confocal immunofluorescence of muscle biopsy from Pt 01 (tibialis anterior post‐treatment) stained with anti‐dystrophin dys1 antibody (which recognizes protein fragment encoded by exons 26–30, green signal) and anti‐laminin‐2 (to delineate muscle fibers, red signal); DAPI identifies nuclei (blue signal). In the left image, only anti‐dystrophin staining is shown. No dystrophin‐positive fibers were observed. Scale bar, 100 μm. Confocal immunofluorescence of muscle biopsy from Pt 02 (tibialis anterior post‐treatment) stained with anti‐dystrophin dys1 antibody (green signal) and anti‐laminin‐2 (red signal); DAPI identifies nuclei (blue signal). In the left image, only anti‐dystrophin staining is shown. Some fiber shows mild and discontinuous dystrophin staining. Scale bar, 100 μm. Immunofluorescence of muscle biopsy from Pt 05 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti‐dystrophin dys2 antibody (which recognizes exons 77–79). The number and intensity of dystrophin‐positive fibers is increased in the post‐treatment biopsy. Scale bar, 80 μm. Immunofluorescence of muscle biopsy from Pt 06 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti‐dystrophin dys2 antibody. Few fibers show scattered dystrophin staining, without obvious differences between pre‐ and post‐treatment samples. Scale bar, 80 μm. Results of Western blot analysis involving dystrophin antibodies dys1, Mandys18, and Manex46e (recognizing respectively exons 26–30, 17–35, and 46), of total protein extracts (20 μg) obtained from post‐treatment biopsy specimens of Pt 01 (tibialis anterior muscle); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. No bands corresponding to full‐length dystrophin were observed. A schematic representation of the deleted portion of the dystrophin and the region recognized by the used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys18 and Manex46e, of total protein extracts (10–20 μg) obtained from pre‐treatment (performed at time of diagnosis) and post‐treatment biopsy specimens of Pt 02 (tibialis anterior muscle); Ct was used as a positive dystrophin control, CtDmd was used as a dystrophin‐negative control. Below, bands corresponding to myosin heavy chain are shown as a loading control. One faint band corresponding to full‐length dystrophin is observed only in the post‐treatment samples with both Mandys18 and Manex46e antibodies. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys106 (recognizing exon 43) and dys1, of total protein extracts (80 μg, following protein concentration by <t>Amicon</t> <t>Ultra‐0.5</t> centrifugal filter devices; Millipore) obtained from pre‐treatment and post‐treatment biopsy specimens of Pt 05 (gastrocnemius); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. Bands corresponding approximately to full‐length dystrophin are observed in pre‐ and post‐treatment samples with both antibodies. However, bands in post‐treatment sample appeared higher in amount. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by the used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys106 and dys1, of total protein extracts (80 μg, following protein concentration by Amicon Ultra‐0.5 centrifugal filter devices, Millipore) obtained from pre‐treatment and post‐treatment biopsy specimens of Pt 06 (gastrocnemius); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. Bands corresponding approximately to full‐length dystrophin are observed in pre‐ and post‐treatment samples with both antibodies. Bands in pre‐treatment sample appeared higher in amount as compared to post‐treatment sample. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by the used antibodies is depicted above the Western blot. Source data are available online for this figure.
    Amicon Ultra 0 5 Centrifugal Filter, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 929 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA amicon ultra centrifugal filters
    Effects of MAB treatment on dystrophin expression Confocal immunofluorescence of muscle biopsy from Pt 01 (tibialis anterior post‐treatment) stained with anti‐dystrophin dys1 antibody (which recognizes protein fragment encoded by exons 26–30, green signal) and anti‐laminin‐2 (to delineate muscle fibers, red signal); DAPI identifies nuclei (blue signal). In the left image, only anti‐dystrophin staining is shown. No dystrophin‐positive fibers were observed. Scale bar, 100 μm. Confocal immunofluorescence of muscle biopsy from Pt 02 (tibialis anterior post‐treatment) stained with anti‐dystrophin dys1 antibody (green signal) and anti‐laminin‐2 (red signal); DAPI identifies nuclei (blue signal). In the left image, only anti‐dystrophin staining is shown. Some fiber shows mild and discontinuous dystrophin staining. Scale bar, 100 μm. Immunofluorescence of muscle biopsy from Pt 05 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti‐dystrophin dys2 antibody (which recognizes exons 77–79). The number and intensity of dystrophin‐positive fibers is increased in the post‐treatment biopsy. Scale bar, 80 μm. Immunofluorescence of muscle biopsy from Pt 06 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti‐dystrophin dys2 antibody. Few fibers show scattered dystrophin staining, without obvious differences between pre‐ and post‐treatment samples. Scale bar, 80 μm. Results of Western blot analysis involving dystrophin antibodies dys1, Mandys18, and Manex46e (recognizing respectively exons 26–30, 17–35, and 46), of total protein extracts (20 μg) obtained from post‐treatment biopsy specimens of Pt 01 (tibialis anterior muscle); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. No bands corresponding to full‐length dystrophin were observed. A schematic representation of the deleted portion of the dystrophin and the region recognized by the used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys18 and Manex46e, of total protein extracts (10–20 μg) obtained from pre‐treatment (performed at time of diagnosis) and post‐treatment biopsy specimens of Pt 02 (tibialis anterior muscle); Ct was used as a positive dystrophin control, CtDmd was used as a dystrophin‐negative control. Below, bands corresponding to myosin heavy chain are shown as a loading control. One faint band corresponding to full‐length dystrophin is observed only in the post‐treatment samples with both Mandys18 and Manex46e antibodies. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys106 (recognizing exon 43) and dys1, of total protein extracts (80 μg, following protein concentration by <t>Amicon</t> <t>Ultra‐0.5</t> centrifugal filter devices; Millipore) obtained from pre‐treatment and post‐treatment biopsy specimens of Pt 05 (gastrocnemius); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. Bands corresponding approximately to full‐length dystrophin are observed in pre‐ and post‐treatment samples with both antibodies. However, bands in post‐treatment sample appeared higher in amount. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by the used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys106 and dys1, of total protein extracts (80 μg, following protein concentration by Amicon Ultra‐0.5 centrifugal filter devices, Millipore) obtained from pre‐treatment and post‐treatment biopsy specimens of Pt 06 (gastrocnemius); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. Bands corresponding approximately to full‐length dystrophin are observed in pre‐ and post‐treatment samples with both antibodies. Bands in pre‐treatment sample appeared higher in amount as compared to post‐treatment sample. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by the used antibodies is depicted above the Western blot. Source data are available online for this figure.
    Amicon Ultra Centrifugal Filters, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 961 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 961 article reviews
    Price from $9.99 to $1999.99
    amicon ultra centrifugal filters - by Bioz Stars, 2020-09
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    Examination of ssDNA_ODN amounts in the concentrate and flow-through after centrifugation of water or 5 M urea solutions containing 10 μ g ssDNA_ODN using Amicon Ultra 30K centrifugal filters. Samples of 15 μ L were analyzed on a 10% denaturing urea-polyacrylamide gel. Lane 1: 300 ng ssDNA_ODN (positive control), Lane 2: concentrate of water solution, Lane 3: flow-through of water solution, Lane 4: concentrate of 5 M urea solution, and Lane 5: flow-through of 5 M urea solution.

    Journal: BioMed Research International

    Article Title: Use of Synthetic Single-Stranded Oligonucleotides as Artificial Test Soiling for Validation of Surgical Instrument Cleaning Processes

    doi: 10.1155/2014/632127

    Figure Lengend Snippet: Examination of ssDNA_ODN amounts in the concentrate and flow-through after centrifugation of water or 5 M urea solutions containing 10 μ g ssDNA_ODN using Amicon Ultra 30K centrifugal filters. Samples of 15 μ L were analyzed on a 10% denaturing urea-polyacrylamide gel. Lane 1: 300 ng ssDNA_ODN (positive control), Lane 2: concentrate of water solution, Lane 3: flow-through of water solution, Lane 4: concentrate of 5 M urea solution, and Lane 5: flow-through of 5 M urea solution.

    Article Snippet: Therefore, ssDNA_ODN in the eluates was concentrated and purified using Amicon Ultra 0.5 mL 30K centrifugal filters (Millipore GmbH, Schwalbach/Ts., Germany) according to the manufacturer's instructions to prevent an effect on qPCR.

    Techniques: Flow Cytometry, Centrifugation, Positive Control

    Comparison of ssDNA_ODN elution efficiency of water or 5 M urea solution at 93°C from the surface of test objects. Using qPCR, detectable amounts of ssDNA_ODN were determined after the elution of immobilized ssDNA_ODN in water or 5 M urea solution ( n = 3). The ssDNA_ODN amount (5 μ g) used for coating of test objects was added to each elution solution. The detected ssDNA_ODN amounts after the Amicon Ultra 30K centrifugal filter purification and concentration were set to 100% (positive controls). The results are presented relative to the positive control as means ± SEM.

    Journal: BioMed Research International

    Article Title: Use of Synthetic Single-Stranded Oligonucleotides as Artificial Test Soiling for Validation of Surgical Instrument Cleaning Processes

    doi: 10.1155/2014/632127

    Figure Lengend Snippet: Comparison of ssDNA_ODN elution efficiency of water or 5 M urea solution at 93°C from the surface of test objects. Using qPCR, detectable amounts of ssDNA_ODN were determined after the elution of immobilized ssDNA_ODN in water or 5 M urea solution ( n = 3). The ssDNA_ODN amount (5 μ g) used for coating of test objects was added to each elution solution. The detected ssDNA_ODN amounts after the Amicon Ultra 30K centrifugal filter purification and concentration were set to 100% (positive controls). The results are presented relative to the positive control as means ± SEM.

    Article Snippet: Therefore, ssDNA_ODN in the eluates was concentrated and purified using Amicon Ultra 0.5 mL 30K centrifugal filters (Millipore GmbH, Schwalbach/Ts., Germany) according to the manufacturer's instructions to prevent an effect on qPCR.

    Techniques: Real-time Polymerase Chain Reaction, Purification, Concentration Assay, Positive Control

    Overview of the quantification of the residual ssDNA_ODN amount on test objects after cleaning in a medical cleaning machine. Stainless steel test objects are coated with a defined amount of ssDNA_ODN and cleaned in a medical cleaning machine. Using 5 M urea solution at 93°C, residual ssDNA_ODN is eluted from the surface of test objects. Eluates are purified and concentrated using Amicon Ultra 30K centrifugal filters. The ssDNA_ODN amount is quantified using quantitative real-time PCR (qPCR).

    Journal: BioMed Research International

    Article Title: Use of Synthetic Single-Stranded Oligonucleotides as Artificial Test Soiling for Validation of Surgical Instrument Cleaning Processes

    doi: 10.1155/2014/632127

    Figure Lengend Snippet: Overview of the quantification of the residual ssDNA_ODN amount on test objects after cleaning in a medical cleaning machine. Stainless steel test objects are coated with a defined amount of ssDNA_ODN and cleaned in a medical cleaning machine. Using 5 M urea solution at 93°C, residual ssDNA_ODN is eluted from the surface of test objects. Eluates are purified and concentrated using Amicon Ultra 30K centrifugal filters. The ssDNA_ODN amount is quantified using quantitative real-time PCR (qPCR).

    Article Snippet: Therefore, ssDNA_ODN in the eluates was concentrated and purified using Amicon Ultra 0.5 mL 30K centrifugal filters (Millipore GmbH, Schwalbach/Ts., Germany) according to the manufacturer's instructions to prevent an effect on qPCR.

    Techniques: Purification, Real-time Polymerase Chain Reaction

    Free S1 NTS binds to the ribosome and interferes with translation of the canonical ompA mRNA. ( A ) Purified S1-depleted 30S ribosomes (30S(-S1)) were incubated in the absence (lanes 1 and 2) or in the presence of FITC labelled S1 NTS (lanes 7 and 8), native protein S1 (lanes 9 and 10) or both (lanes 11 and 12). Likewise, FITC labelled S1 NTS (lanes 3 and 4) or native S1 (lanes 5 and 6) were incubated in the absence of ribosomes. Before (input; lanes 1, 3, 5, 7, 9 and 11) and after ultrafiltration using 100 kDa MWCO Amicon concentrators (Millipore) samples were taken and the presence of the respective proteins and the S1 NTS peptide in the ribosome fraction (ribosome fraction; lanes 2, 4, 6, 8, 10 and 12) was determined by SDS-PAGE. ( B ) In vitro translation of ompA mRNA in the absence (lane 1) or in the presence of a 10- or 50-fold molar excess over ribosomes of S1 NTD (lanes 2 and 3), S1 D1 (lanes 4 and 5) or S1 NTS (lanes 6 and 7), respectively. The assay was performed in triplicate and one representative autoradiograph is shown. Graph representing the quantification of three independent assays is given below. Error bars represent the standard deviation of the mean.

    Journal: Nucleic Acids Research

    Article Title: Structural basis for the interaction of protein S1 with the Escherichia coli ribosome

    doi: 10.1093/nar/gku1314

    Figure Lengend Snippet: Free S1 NTS binds to the ribosome and interferes with translation of the canonical ompA mRNA. ( A ) Purified S1-depleted 30S ribosomes (30S(-S1)) were incubated in the absence (lanes 1 and 2) or in the presence of FITC labelled S1 NTS (lanes 7 and 8), native protein S1 (lanes 9 and 10) or both (lanes 11 and 12). Likewise, FITC labelled S1 NTS (lanes 3 and 4) or native S1 (lanes 5 and 6) were incubated in the absence of ribosomes. Before (input; lanes 1, 3, 5, 7, 9 and 11) and after ultrafiltration using 100 kDa MWCO Amicon concentrators (Millipore) samples were taken and the presence of the respective proteins and the S1 NTS peptide in the ribosome fraction (ribosome fraction; lanes 2, 4, 6, 8, 10 and 12) was determined by SDS-PAGE. ( B ) In vitro translation of ompA mRNA in the absence (lane 1) or in the presence of a 10- or 50-fold molar excess over ribosomes of S1 NTD (lanes 2 and 3), S1 D1 (lanes 4 and 5) or S1 NTS (lanes 6 and 7), respectively. The assay was performed in triplicate and one representative autoradiograph is shown. Graph representing the quantification of three independent assays is given below. Error bars represent the standard deviation of the mean.

    Article Snippet: The fractions containing the chimeric protein were concentrated with an Amicon ultra centrifugal filter unit (MWCO of 30 kDa; Millipore).

    Techniques: Purification, Incubation, SDS Page, In Vitro, Autoradiography, Standard Deviation

    Met-RANTES inhibits regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5)-induced matrix metalloproteinase (MMP)-1 and MMP-13 expression in rheumatoid arthritis synovial fibroblasts (RASFs). (A,B) Effect of Met-RANTES on RANTES/CCL5-induced MMP-1 and MMP-13 mRNA and (C,D) at protein levels was determined using qRT-PCR and ELISA, respectively. RASFs were pretreated with Met-RANTES (50, 100, and 200 ng/ml) for 30 min followed by stimulation with RANTES/CCL5 (100 ng/ml) for 24 h. (E,F) Human RASFs were cultured in three-dimensional (3D) micromass for 21 days to form synovial tissue-like architect, followed by pretreatment with or without Met-RANTES for 30 min and then stimulation with RANTES (100 ng/ml) for 24 h. Interleukin (IL)-1β (10 ng/ml) 24 h stimulated RASF micromass were used as the positive control. Conditioned media was concentrated using Amicon ® Ultra Centrifugal filters (Millipore) and MMP-1 and MMP-13 production was analyzed using Western immunoblotting. Densitometry was performed to determine the relative changes. Values are represented as mean ± SE from three to four independent experiments using cells from different donors. # p

    Journal: Frontiers in Immunology

    Article Title: RANTES/CCL5 Induces Collagen Degradation by Activating MMP-1 and MMP-13 Expression in Human Rheumatoid Arthritis Synovial Fibroblasts

    doi: 10.3389/fimmu.2017.01341

    Figure Lengend Snippet: Met-RANTES inhibits regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5)-induced matrix metalloproteinase (MMP)-1 and MMP-13 expression in rheumatoid arthritis synovial fibroblasts (RASFs). (A,B) Effect of Met-RANTES on RANTES/CCL5-induced MMP-1 and MMP-13 mRNA and (C,D) at protein levels was determined using qRT-PCR and ELISA, respectively. RASFs were pretreated with Met-RANTES (50, 100, and 200 ng/ml) for 30 min followed by stimulation with RANTES/CCL5 (100 ng/ml) for 24 h. (E,F) Human RASFs were cultured in three-dimensional (3D) micromass for 21 days to form synovial tissue-like architect, followed by pretreatment with or without Met-RANTES for 30 min and then stimulation with RANTES (100 ng/ml) for 24 h. Interleukin (IL)-1β (10 ng/ml) 24 h stimulated RASF micromass were used as the positive control. Conditioned media was concentrated using Amicon ® Ultra Centrifugal filters (Millipore) and MMP-1 and MMP-13 production was analyzed using Western immunoblotting. Densitometry was performed to determine the relative changes. Values are represented as mean ± SE from three to four independent experiments using cells from different donors. # p

    Article Snippet: For some experiments, RASFs were pretreated with the inhibitor of p38 (SB203980; 10 µM), ERK (PD98059; 10 µM), JNK (SP600125; 10 µM), NF-KB (PDTC; 200 µM), or PKCδ (Rottlerin; 10 µM) for 2 h followed by RANTES/CCL5 treatment for 24 h. Culture supernatants were concentrated using Amicon® Ultra centrifugal filters (Millipore) and MMP-1 and MMP-13 expression was determined using Western immunoblotting.

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Positive Control, Western Blot

    Regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5) facilitates matrix metalloproteinases (MMPs)-mediated collagen degradation in rheumatoid arthritis (RA). (A) RA synovial fibroblasts (RASFs) from five donors were stimulated with RANTES/CCL5 (100 ng/ml) and IL-1β (10 ng/ml) for 24 h. Condition media was placed onto a 96-well plate coated with type I collagen film and incubated for 24 h. Plates were stained with Coomassie R250 analyzed by ChemiDoc™ Imager. (B) Optical density (570 nm) of the RANTES/CCL5 and IL-1β-stimulated samples compared to control is shown. (C) Condition media from the same treatment was concentrated using Amicon 10K ultra 0.5 centrifugal filters and analyzed for type I collagen by Western blotting. (D) Condition media from RANTES/CCL-5 or IL-1β stimulated RASFs, was applied on collagen coated 96-well plates for 8 and 48 h. Digested collagen was collected and CD spectroscopy was performed using a Jasco J-815 spectropolarimeter. (E) RASFs were pretreated with GM6001 (20 µM) for 2 h and then stimulated with RANTES/CCL5 (100 ng/ml) or IL-1β (10 ng/ml). Collagen type I plates were exposed for 24 h, media aspirated, and then stained with Coomassie R250 and analyzed as described in Section “ Materials and Methods .” Densitometry was performed to show the relative changes. Values are represented as MEAN ± SE from experiments performed using cells from four to five donors. * p

    Journal: Frontiers in Immunology

    Article Title: RANTES/CCL5 Induces Collagen Degradation by Activating MMP-1 and MMP-13 Expression in Human Rheumatoid Arthritis Synovial Fibroblasts

    doi: 10.3389/fimmu.2017.01341

    Figure Lengend Snippet: Regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5) facilitates matrix metalloproteinases (MMPs)-mediated collagen degradation in rheumatoid arthritis (RA). (A) RA synovial fibroblasts (RASFs) from five donors were stimulated with RANTES/CCL5 (100 ng/ml) and IL-1β (10 ng/ml) for 24 h. Condition media was placed onto a 96-well plate coated with type I collagen film and incubated for 24 h. Plates were stained with Coomassie R250 analyzed by ChemiDoc™ Imager. (B) Optical density (570 nm) of the RANTES/CCL5 and IL-1β-stimulated samples compared to control is shown. (C) Condition media from the same treatment was concentrated using Amicon 10K ultra 0.5 centrifugal filters and analyzed for type I collagen by Western blotting. (D) Condition media from RANTES/CCL-5 or IL-1β stimulated RASFs, was applied on collagen coated 96-well plates for 8 and 48 h. Digested collagen was collected and CD spectroscopy was performed using a Jasco J-815 spectropolarimeter. (E) RASFs were pretreated with GM6001 (20 µM) for 2 h and then stimulated with RANTES/CCL5 (100 ng/ml) or IL-1β (10 ng/ml). Collagen type I plates were exposed for 24 h, media aspirated, and then stained with Coomassie R250 and analyzed as described in Section “ Materials and Methods .” Densitometry was performed to show the relative changes. Values are represented as MEAN ± SE from experiments performed using cells from four to five donors. * p

    Article Snippet: For some experiments, RASFs were pretreated with the inhibitor of p38 (SB203980; 10 µM), ERK (PD98059; 10 µM), JNK (SP600125; 10 µM), NF-KB (PDTC; 200 µM), or PKCδ (Rottlerin; 10 µM) for 2 h followed by RANTES/CCL5 treatment for 24 h. Culture supernatants were concentrated using Amicon® Ultra centrifugal filters (Millipore) and MMP-1 and MMP-13 expression was determined using Western immunoblotting.

    Techniques: Activation Assay, Incubation, Staining, Western Blot, Spectroscopy

    Regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5) induces matrix metalloproteinase (MMP)-1 and MMP-13 expression in rheumatoid arthritis synovial fibroblasts (RASFs). (A) Effect of RANTES/CCL5 on MMP-1 and MMP-13 mRNA and (B) protein expression was studied in RASFs. (C,D) Human OASFs and NLSFs were treated with different concentrations of recombinant RANTES/CCL5 (20, 50, and 100 ng/ml) and MMP-1 and MMP-13 expression was analyzed using Western immunoblotting, respectively. Gels described for (B–D) were stained with Commassie Blue stain to ensure equal loading of the supernatants. (E) Effect of RANTES/CCL5 on collagenase activity in RASFs. RASFs were treated with different concentration (20, 50, and 100 ng/ml) of RANTES/CCL5 for 24 h and conditioned media was concentrated using Amicon ® Ultra Centrifugal filters (Millipore) and resolved on polyacrylamide gel loaded with collagen. Zymography image of digested regions representing MMPs activity is shown. Fold changes on developed zymograms was determined using densitometric analysis. (F) Effect of RANTES/CCL5 on the cell viability of RASFs was determined using MTT assay. RASFs were treated with RANTES/CCL5 (20, 50, and 100 ng/ml) for 24 h. Cell viability of cultured RASFs in the presence of RANTES/CCL5 was measured by optical densities at 570 nm. Values are represented as mean ± SE from three to four independent experiments using cells from different donors under similar conditions. * p

    Journal: Frontiers in Immunology

    Article Title: RANTES/CCL5 Induces Collagen Degradation by Activating MMP-1 and MMP-13 Expression in Human Rheumatoid Arthritis Synovial Fibroblasts

    doi: 10.3389/fimmu.2017.01341

    Figure Lengend Snippet: Regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5) induces matrix metalloproteinase (MMP)-1 and MMP-13 expression in rheumatoid arthritis synovial fibroblasts (RASFs). (A) Effect of RANTES/CCL5 on MMP-1 and MMP-13 mRNA and (B) protein expression was studied in RASFs. (C,D) Human OASFs and NLSFs were treated with different concentrations of recombinant RANTES/CCL5 (20, 50, and 100 ng/ml) and MMP-1 and MMP-13 expression was analyzed using Western immunoblotting, respectively. Gels described for (B–D) were stained with Commassie Blue stain to ensure equal loading of the supernatants. (E) Effect of RANTES/CCL5 on collagenase activity in RASFs. RASFs were treated with different concentration (20, 50, and 100 ng/ml) of RANTES/CCL5 for 24 h and conditioned media was concentrated using Amicon ® Ultra Centrifugal filters (Millipore) and resolved on polyacrylamide gel loaded with collagen. Zymography image of digested regions representing MMPs activity is shown. Fold changes on developed zymograms was determined using densitometric analysis. (F) Effect of RANTES/CCL5 on the cell viability of RASFs was determined using MTT assay. RASFs were treated with RANTES/CCL5 (20, 50, and 100 ng/ml) for 24 h. Cell viability of cultured RASFs in the presence of RANTES/CCL5 was measured by optical densities at 570 nm. Values are represented as mean ± SE from three to four independent experiments using cells from different donors under similar conditions. * p

    Article Snippet: For some experiments, RASFs were pretreated with the inhibitor of p38 (SB203980; 10 µM), ERK (PD98059; 10 µM), JNK (SP600125; 10 µM), NF-KB (PDTC; 200 µM), or PKCδ (Rottlerin; 10 µM) for 2 h followed by RANTES/CCL5 treatment for 24 h. Culture supernatants were concentrated using Amicon® Ultra centrifugal filters (Millipore) and MMP-1 and MMP-13 expression was determined using Western immunoblotting.

    Techniques: Activation Assay, Expressing, Recombinant, Western Blot, Staining, Activity Assay, Concentration Assay, Zymography, MTT Assay, Cell Culture

    CAA standard series in urine analysed with the UCP-LF concentration assays. The amount of urine analysed per strip increased from 10, 250 to 2000 μL, respectively for the UCAA10, −250 and −2000 assay. The UCAA250 and UCA2000 require concentration of the TCA-supernatant using Amicon Centrifugal Filter Devices Ultra-0.5 and Ultra-4, respectively.

    Journal: Parasites & Vectors

    Article Title: Improved sensitivity of the urine CAA lateral-flow assay for diagnosing active Schistosoma infections by using larger sample volumes

    doi: 10.1186/s13071-015-0857-7

    Figure Lengend Snippet: CAA standard series in urine analysed with the UCP-LF concentration assays. The amount of urine analysed per strip increased from 10, 250 to 2000 μL, respectively for the UCAA10, −250 and −2000 assay. The UCAA250 and UCA2000 require concentration of the TCA-supernatant using Amicon Centrifugal Filter Devices Ultra-0.5 and Ultra-4, respectively.

    Article Snippet: This step involves the concentration of the TCA-soluble fraction of urine samples using Amicon Ultra Centrifugal Filter Devices (Millipore Corp.), allowing larger sample input, increasing the volume from 10 μL urine to 250 and 2000 μL, or even 7500 μL.

    Techniques: Cellular Antioxidant Activity Assay, Concentration Assay, Stripping Membranes

    Effect of increased sample input on the LLOD of the UCP-LF CAA assay. AWA-TCA standard series in urine (CAA concentration indicated in upper panel), analysed in triplicate with the UCP-LF wet assay format. Spiked urine was extracted with 1 volume 4% (v/v) TCA, the resulting TCA-supernatant was either tested directly with the UCAA10 assay or first concentrated using 0.5, 4, and 15 mL Amicon centrifugal filter devices. Arrows in the lower panel (a blow up of the y-axis) indicate the QC cutoff threshold for the different assays; left to right: UCAA10, −250, −2000 and −7500. Error bars indicate 1 standard deviation (n = 3).

    Journal: Parasites & Vectors

    Article Title: Improved sensitivity of the urine CAA lateral-flow assay for diagnosing active Schistosoma infections by using larger sample volumes

    doi: 10.1186/s13071-015-0857-7

    Figure Lengend Snippet: Effect of increased sample input on the LLOD of the UCP-LF CAA assay. AWA-TCA standard series in urine (CAA concentration indicated in upper panel), analysed in triplicate with the UCP-LF wet assay format. Spiked urine was extracted with 1 volume 4% (v/v) TCA, the resulting TCA-supernatant was either tested directly with the UCAA10 assay or first concentrated using 0.5, 4, and 15 mL Amicon centrifugal filter devices. Arrows in the lower panel (a blow up of the y-axis) indicate the QC cutoff threshold for the different assays; left to right: UCAA10, −250, −2000 and −7500. Error bars indicate 1 standard deviation (n = 3).

    Article Snippet: This step involves the concentration of the TCA-soluble fraction of urine samples using Amicon Ultra Centrifugal Filter Devices (Millipore Corp.), allowing larger sample input, increasing the volume from 10 μL urine to 250 and 2000 μL, or even 7500 μL.

    Techniques: Cellular Antioxidant Activity Assay, Concentration Assay, Standard Deviation

    Inflammatory component produced by Vero cells during DENV propagation is responsible for IL-1β induction. (A) Schematic depicting the different depletion and control fractions for depleting antibody-bound virions from DIV crude supernatant by using magnetic protein G beads. (B) Immunoblot for human IgG Fc, and two DENV structural proteins, E protein and prM, to assess efficiency of antibody-mediated DENV depletion from DIV crude supernatant. “*” denotes all 3 antibodies were used in combination at 1 μg/ml each. 3 μg/ml of each antibody was used for individual antibody conditions. (C) Control supernatants (Lane C) and residual supernatants (Lane R) from panel B were inoculated onto mobilized monocytes. Monocyte supernatants were collected at 24 hpi, and secreted IL-1β was measured by ELISA. (D) Mock and DIV crude supernatants were incubated with PBS or anti-DENV NS1 antibody prior to addition of protein G beads and subsequent depletion. Top: Residual supernatants (R) and bead-bound fractions (B) were assessed for efficiency of NS1 depletion by immunoblot. Bottom: Residual supernatants were inoculated onto mobilized monocytes. At 4 hpi, monocyte supernatants were collected and assessed for secreted IL-1β. (E) Mock and DIV crude supernatants were incubated with PBS, DNase, or Riboshredder prior to inoculation onto mobilized monocytes. At 4 hpi, monocyte supernatants were collected and assessed for secreted IL-1β. (F) Mobilized monocytes were inoculated with mock supernatant, live DIV crude supernatant, or DIV crude supernatant that had been incubated for 30 minutes at 56°C to heat inactivate (HI) it. At 24 hpi, monocyte supernatants were collected and assessed for secreted IL-1β. (G) DIV crude supernatant was centrifuged in an Amicon centrifugal filtration unit with a 100-kDa molecular-weight cutoff to generate fractions smaller and larger than 100 kDa. Fractions were brought to equal volumes and inoculated onto mobilized monocytes. At 24 hpi, monocyte supernatants were collected, and IL-1β secretion was measured. Tests used: Two-Way ANOVA with Bonferroni’s post-test (D), One-Way ANOVA with Dunnett’s post-test (F).

    Journal: PLoS ONE

    Article Title: Source and Purity of Dengue-Viral Preparations Impact Requirement for Enhancing Antibody to Induce Elevated IL-1β Secretion: A Primary Human Monocyte Model

    doi: 10.1371/journal.pone.0136708

    Figure Lengend Snippet: Inflammatory component produced by Vero cells during DENV propagation is responsible for IL-1β induction. (A) Schematic depicting the different depletion and control fractions for depleting antibody-bound virions from DIV crude supernatant by using magnetic protein G beads. (B) Immunoblot for human IgG Fc, and two DENV structural proteins, E protein and prM, to assess efficiency of antibody-mediated DENV depletion from DIV crude supernatant. “*” denotes all 3 antibodies were used in combination at 1 μg/ml each. 3 μg/ml of each antibody was used for individual antibody conditions. (C) Control supernatants (Lane C) and residual supernatants (Lane R) from panel B were inoculated onto mobilized monocytes. Monocyte supernatants were collected at 24 hpi, and secreted IL-1β was measured by ELISA. (D) Mock and DIV crude supernatants were incubated with PBS or anti-DENV NS1 antibody prior to addition of protein G beads and subsequent depletion. Top: Residual supernatants (R) and bead-bound fractions (B) were assessed for efficiency of NS1 depletion by immunoblot. Bottom: Residual supernatants were inoculated onto mobilized monocytes. At 4 hpi, monocyte supernatants were collected and assessed for secreted IL-1β. (E) Mock and DIV crude supernatants were incubated with PBS, DNase, or Riboshredder prior to inoculation onto mobilized monocytes. At 4 hpi, monocyte supernatants were collected and assessed for secreted IL-1β. (F) Mobilized monocytes were inoculated with mock supernatant, live DIV crude supernatant, or DIV crude supernatant that had been incubated for 30 minutes at 56°C to heat inactivate (HI) it. At 24 hpi, monocyte supernatants were collected and assessed for secreted IL-1β. (G) DIV crude supernatant was centrifuged in an Amicon centrifugal filtration unit with a 100-kDa molecular-weight cutoff to generate fractions smaller and larger than 100 kDa. Fractions were brought to equal volumes and inoculated onto mobilized monocytes. At 24 hpi, monocyte supernatants were collected, and IL-1β secretion was measured. Tests used: Two-Way ANOVA with Bonferroni’s post-test (D), One-Way ANOVA with Dunnett’s post-test (F).

    Article Snippet: Briefly, crude supernatants from DENV-infected Vero cells were concentrated by centrifugation at 1500 x g for 20–25 minutes in 15 ml Millipore Amicon Centrifugal Filter Units with a 100-kDa cutoff, allowing components < 100 kDa to pass through but not virus.

    Techniques: Produced, Enzyme-linked Immunosorbent Assay, Incubation, Filtration, Molecular Weight

    MDA-MB-231 aggressive breast cancer cells process and secrete PDGF-C into culture medium. A and B , the PDGF-C protein levels in various cancer cell culture supernatants were subjected to Western blot analysis. After 48 h of incubation in serum-free medium, supernatants from MDA-MB-231, MCF-7, MeWo, A549, and HT-29 cells were concentrated using a 10-K Amicon Ultra-15 centrifugal filter. Each concentration was subjected to Western blot analysis using anti-PDGF-C antibody under reducing ( A ) and non-reducing ( B ) conditions. The molecular mass (kDa) is indicated on the left side. C , real-time RT-PCR quantification of PDGF-C mRNA expression in various cancer cell lines. GAPDH is used for normalization of levels. Individual error bars represent the mean ( bars ) ± S.D. of triplicates. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation *

    doi: 10.1074/jbc.M113.508994

    Figure Lengend Snippet: MDA-MB-231 aggressive breast cancer cells process and secrete PDGF-C into culture medium. A and B , the PDGF-C protein levels in various cancer cell culture supernatants were subjected to Western blot analysis. After 48 h of incubation in serum-free medium, supernatants from MDA-MB-231, MCF-7, MeWo, A549, and HT-29 cells were concentrated using a 10-K Amicon Ultra-15 centrifugal filter. Each concentration was subjected to Western blot analysis using anti-PDGF-C antibody under reducing ( A ) and non-reducing ( B ) conditions. The molecular mass (kDa) is indicated on the left side. C , real-time RT-PCR quantification of PDGF-C mRNA expression in various cancer cell lines. GAPDH is used for normalization of levels. Individual error bars represent the mean ( bars ) ± S.D. of triplicates. ***, p

    Article Snippet: After 48 h, culture supernatants containing cancer cell-secreted proteins were collected, and 13–15 ml media were concentrated in a swinging bucket rotor at 3900 × g at 25 °C for 18–20 min using a 10-K Amicon Ultra-15 centrifugal filter (Millipore).

    Techniques: Multiple Displacement Amplification, Cell Culture, Western Blot, Incubation, Concentration Assay, Quantitative RT-PCR, Expressing

    The specific blockade of TβRI kinase ablated Smad2 activation and dramatically affected the induction of the EMT markers, vimentin and MMPs by FK506. HK-2 cells were cultured on 6-well plates and treated with vehicle control, 5 µM FK506 with or without 500 nM of a TGFβRI/ALK5 kinase inhibitor V (SD-208). ( A ) Cells were incubated for 48 h with SD-208 concentrations ranging from 0–20μM. LDH and resazurin assays were conducted to assess SD-208 inhibitor toxicity; ( B ) Whole cell protein was extracted at 48 h with RIPA buffer. Equal amounts of protein were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose and indirectly probed for vimentin and fibronectin using a mAb and ECL detection system. Whole Smad2 and GAPDH was used as a loading control. A representative blot is shown from three independent experiments. Densitometry is shown for the replicate blots; ( C ) Cell culture supernatants were harvested, centrifuged to remove cellular debris and concentrated using Amicon Ultra-4 Centrifugal Filter Devices (Millipore). Gelatin substrate zymography was used to separate and assess MMP-9 and MMP-2 activity. Clear areas represent areas of gelatinolytic activity. A representative zymogram is shown from three independent experiments. ** and *** indicates different levels of significant compared to time-matched control; * , ** and *** indicates varying levels of significance compared to treatment + inhibitor.

    Journal: Journal of Clinical Medicine

    Article Title: Tacrolimus Modulates TGF-β Signaling to Induce Epithelial-Mesenchymal Transition in Human Renal Proximal Tubule Epithelial Cells

    doi: 10.3390/jcm5050050

    Figure Lengend Snippet: The specific blockade of TβRI kinase ablated Smad2 activation and dramatically affected the induction of the EMT markers, vimentin and MMPs by FK506. HK-2 cells were cultured on 6-well plates and treated with vehicle control, 5 µM FK506 with or without 500 nM of a TGFβRI/ALK5 kinase inhibitor V (SD-208). ( A ) Cells were incubated for 48 h with SD-208 concentrations ranging from 0–20μM. LDH and resazurin assays were conducted to assess SD-208 inhibitor toxicity; ( B ) Whole cell protein was extracted at 48 h with RIPA buffer. Equal amounts of protein were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose and indirectly probed for vimentin and fibronectin using a mAb and ECL detection system. Whole Smad2 and GAPDH was used as a loading control. A representative blot is shown from three independent experiments. Densitometry is shown for the replicate blots; ( C ) Cell culture supernatants were harvested, centrifuged to remove cellular debris and concentrated using Amicon Ultra-4 Centrifugal Filter Devices (Millipore). Gelatin substrate zymography was used to separate and assess MMP-9 and MMP-2 activity. Clear areas represent areas of gelatinolytic activity. A representative zymogram is shown from three independent experiments. ** and *** indicates different levels of significant compared to time-matched control; * , ** and *** indicates varying levels of significance compared to treatment + inhibitor.

    Article Snippet: Gelatin Zymography: Cell supernatants were concentrated using Amicon Ultra-4 Centrifugal Filter Devices (Millipore, Billerica, MA, USA).

    Techniques: Activation Assay, Cell Culture, Incubation, SDS Page, Electrophoresis, Zymography, Activity Assay

    MS rats show increased insulin binding to plasmatic proteins . (A) Western blot from plasma of control rats (lane 1, 2) and MS rats (3, 4, 5) that were previously separated using different Amicon centrifugal filters and immunoprecipitated with an insulin receptor (alpha-IR) in a native gel, stained with an anti-insulin antibody. Observe that insulin is present in different molecular weight bands, Section “ Materials and Methods .” (B) Rats with metabolic syndrome (MS) show increased insulin levels when compared to control rats (C). (C) MS rats display higher insulin levels in the plasmatic fraction containing proteins above 30 kDa, suggesting higher insulin binding to plasmatic proteins. Bars represent mean ± SEM of control ( n = 12) and MS rats ( n = 7), * p

    Journal: Frontiers in Endocrinology

    Article Title: Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes

    doi: 10.3389/fendo.2014.00095

    Figure Lengend Snippet: MS rats show increased insulin binding to plasmatic proteins . (A) Western blot from plasma of control rats (lane 1, 2) and MS rats (3, 4, 5) that were previously separated using different Amicon centrifugal filters and immunoprecipitated with an insulin receptor (alpha-IR) in a native gel, stained with an anti-insulin antibody. Observe that insulin is present in different molecular weight bands, Section “ Materials and Methods .” (B) Rats with metabolic syndrome (MS) show increased insulin levels when compared to control rats (C). (C) MS rats display higher insulin levels in the plasmatic fraction containing proteins above 30 kDa, suggesting higher insulin binding to plasmatic proteins. Bars represent mean ± SEM of control ( n = 12) and MS rats ( n = 7), * p

    Article Snippet: Purification procedures Each run consisted of 1 ml of samples from hepatocyte culture supernatants that were previously concentrated in Amicon centrifugal filters with a cutoff of 30 kDa at 2150 × g and 4°C for 10 min. Then, 50 mg of protein (50% of column capacity) were resuspended in charge buffer (20 mmol l−1 Tris–HCl, pH 7.4, and 500 mM NaCl), filtered with 0.22 μm Millipore membrane filter (Millipore, Billerica, MA, USA), and injected onto a concanavalin-A affinity chromatography column.

    Techniques: Mass Spectrometry, Binding Assay, Western Blot, Immunoprecipitation, Staining, Molecular Weight

    The active ingredient/s within KOSR reside in its high molecular weight fraction. (A) Samples of KOSR were filtered using Amicon Ultra filters (Millipore) of three different molecular weight cutoffs (MWCO), so that only molecules with molecular weights (MW) below the filters' MWCOs could go through them. For each filter, two fractions were obtained corresponding to the low and high MW fractions. The latter fractions were obtained by resuspending the molecules retained by the filters in a volume of D-PBS equal to the original KOSR volume. (B) The six fractions obtained in this manner were analyzed by SDS-PAGE to ensure that filters were working properly. Coomassie blue staining of the gel revealed that filters were indeed preventing high MW components to go through them. Lipid-rich BSA is the most abundant protein component in KOSR by a factor of about 1000 times and this is why no other proteins are seen in the gel. (C) HUES7 cells were cultured for five passages in N2/B27-CDM+15% (V∶V) of each of the six fractions. After this period of time cells were fixed and stained for ALP activity. As seen in the top panel for the > 50 kDa fraction, all high MW fractions were able to maintain widespread ALP expression whereas, as shown in the bottom panel for the

    Journal: PLoS ONE

    Article Title: Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal

    doi: 10.1371/journal.pone.0001384

    Figure Lengend Snippet: The active ingredient/s within KOSR reside in its high molecular weight fraction. (A) Samples of KOSR were filtered using Amicon Ultra filters (Millipore) of three different molecular weight cutoffs (MWCO), so that only molecules with molecular weights (MW) below the filters' MWCOs could go through them. For each filter, two fractions were obtained corresponding to the low and high MW fractions. The latter fractions were obtained by resuspending the molecules retained by the filters in a volume of D-PBS equal to the original KOSR volume. (B) The six fractions obtained in this manner were analyzed by SDS-PAGE to ensure that filters were working properly. Coomassie blue staining of the gel revealed that filters were indeed preventing high MW components to go through them. Lipid-rich BSA is the most abundant protein component in KOSR by a factor of about 1000 times and this is why no other proteins are seen in the gel. (C) HUES7 cells were cultured for five passages in N2/B27-CDM+15% (V∶V) of each of the six fractions. After this period of time cells were fixed and stained for ALP activity. As seen in the top panel for the > 50 kDa fraction, all high MW fractions were able to maintain widespread ALP expression whereas, as shown in the bottom panel for the

    Article Snippet: In order to find out whether the active ingredient/s within KOSR are large or small molecules, we passed samples of KOSR through Amicon Ultra filters (Millipore) with molecular weight cutoffs (MWCO) of 10, 30 and 50 kDa ( ).

    Techniques: Molecular Weight, SDS Page, Staining, Cell Culture, ALP Assay, Activity Assay, Expressing

    The profile of sHA fragments contained in TIF from MTLy (A) and MT-450 (B) tumours. Tumour interstitial fluid from multiple MT-450 or MTLy tumours was pooled and subjected to ultracentrifugal filtration through 10-kDa Amicon filters. The filtrates were concentrated in a Speed Vac, then applied to a Bio Gel P10 size-exclusion chromatography column (3.5 × 115 cm) and eluted with 0.3 M sodium phosphate buffer (pH 5.3). The HA concentrations in the eluted 3 ml fractions were determined by the HA ELISA-like assay. The column was calibrated by fractionating a partial HA digest under identical conditions, then determining the size of the HA oligosaccharides in the fractions using FACE analysis. The region in which HA fragments of a specific length were eluted is indicated in the diagram.

    Journal: British Journal of Cancer

    Article Title: Accumulation of small hyaluronan oligosaccharides in tumour interstitial fluid correlates with lymphatic invasion and lymph node metastasis

    doi: 10.1038/bjc.2014.332

    Figure Lengend Snippet: The profile of sHA fragments contained in TIF from MTLy (A) and MT-450 (B) tumours. Tumour interstitial fluid from multiple MT-450 or MTLy tumours was pooled and subjected to ultracentrifugal filtration through 10-kDa Amicon filters. The filtrates were concentrated in a Speed Vac, then applied to a Bio Gel P10 size-exclusion chromatography column (3.5 × 115 cm) and eluted with 0.3 M sodium phosphate buffer (pH 5.3). The HA concentrations in the eluted 3 ml fractions were determined by the HA ELISA-like assay. The column was calibrated by fractionating a partial HA digest under identical conditions, then determining the size of the HA oligosaccharides in the fractions using FACE analysis. The region in which HA fragments of a specific length were eluted is indicated in the diagram.

    Article Snippet: To separate sHA from HMW-HA, interstitial fluid was centrifuged through Amicon Ultracentrifugal Filters with a molecular weight cutoff (MWCO) of 10 kDa (Millipore, Billerica, MA, USA).

    Techniques: Filtration, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay

    Effects of MAB treatment on dystrophin expression Confocal immunofluorescence of muscle biopsy from Pt 01 (tibialis anterior post‐treatment) stained with anti‐dystrophin dys1 antibody (which recognizes protein fragment encoded by exons 26–30, green signal) and anti‐laminin‐2 (to delineate muscle fibers, red signal); DAPI identifies nuclei (blue signal). In the left image, only anti‐dystrophin staining is shown. No dystrophin‐positive fibers were observed. Scale bar, 100 μm. Confocal immunofluorescence of muscle biopsy from Pt 02 (tibialis anterior post‐treatment) stained with anti‐dystrophin dys1 antibody (green signal) and anti‐laminin‐2 (red signal); DAPI identifies nuclei (blue signal). In the left image, only anti‐dystrophin staining is shown. Some fiber shows mild and discontinuous dystrophin staining. Scale bar, 100 μm. Immunofluorescence of muscle biopsy from Pt 05 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti‐dystrophin dys2 antibody (which recognizes exons 77–79). The number and intensity of dystrophin‐positive fibers is increased in the post‐treatment biopsy. Scale bar, 80 μm. Immunofluorescence of muscle biopsy from Pt 06 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti‐dystrophin dys2 antibody. Few fibers show scattered dystrophin staining, without obvious differences between pre‐ and post‐treatment samples. Scale bar, 80 μm. Results of Western blot analysis involving dystrophin antibodies dys1, Mandys18, and Manex46e (recognizing respectively exons 26–30, 17–35, and 46), of total protein extracts (20 μg) obtained from post‐treatment biopsy specimens of Pt 01 (tibialis anterior muscle); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. No bands corresponding to full‐length dystrophin were observed. A schematic representation of the deleted portion of the dystrophin and the region recognized by the used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys18 and Manex46e, of total protein extracts (10–20 μg) obtained from pre‐treatment (performed at time of diagnosis) and post‐treatment biopsy specimens of Pt 02 (tibialis anterior muscle); Ct was used as a positive dystrophin control, CtDmd was used as a dystrophin‐negative control. Below, bands corresponding to myosin heavy chain are shown as a loading control. One faint band corresponding to full‐length dystrophin is observed only in the post‐treatment samples with both Mandys18 and Manex46e antibodies. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys106 (recognizing exon 43) and dys1, of total protein extracts (80 μg, following protein concentration by Amicon Ultra‐0.5 centrifugal filter devices; Millipore) obtained from pre‐treatment and post‐treatment biopsy specimens of Pt 05 (gastrocnemius); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. Bands corresponding approximately to full‐length dystrophin are observed in pre‐ and post‐treatment samples with both antibodies. However, bands in post‐treatment sample appeared higher in amount. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by the used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys106 and dys1, of total protein extracts (80 μg, following protein concentration by Amicon Ultra‐0.5 centrifugal filter devices, Millipore) obtained from pre‐treatment and post‐treatment biopsy specimens of Pt 06 (gastrocnemius); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. Bands corresponding approximately to full‐length dystrophin are observed in pre‐ and post‐treatment samples with both antibodies. Bands in pre‐treatment sample appeared higher in amount as compared to post‐treatment sample. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by the used antibodies is depicted above the Western blot. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Intra‐arterial transplantation of HLA‐matched donor mesoangioblasts in Duchenne muscular dystrophy

    doi: 10.15252/emmm.201505636

    Figure Lengend Snippet: Effects of MAB treatment on dystrophin expression Confocal immunofluorescence of muscle biopsy from Pt 01 (tibialis anterior post‐treatment) stained with anti‐dystrophin dys1 antibody (which recognizes protein fragment encoded by exons 26–30, green signal) and anti‐laminin‐2 (to delineate muscle fibers, red signal); DAPI identifies nuclei (blue signal). In the left image, only anti‐dystrophin staining is shown. No dystrophin‐positive fibers were observed. Scale bar, 100 μm. Confocal immunofluorescence of muscle biopsy from Pt 02 (tibialis anterior post‐treatment) stained with anti‐dystrophin dys1 antibody (green signal) and anti‐laminin‐2 (red signal); DAPI identifies nuclei (blue signal). In the left image, only anti‐dystrophin staining is shown. Some fiber shows mild and discontinuous dystrophin staining. Scale bar, 100 μm. Immunofluorescence of muscle biopsy from Pt 05 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti‐dystrophin dys2 antibody (which recognizes exons 77–79). The number and intensity of dystrophin‐positive fibers is increased in the post‐treatment biopsy. Scale bar, 80 μm. Immunofluorescence of muscle biopsy from Pt 06 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti‐dystrophin dys2 antibody. Few fibers show scattered dystrophin staining, without obvious differences between pre‐ and post‐treatment samples. Scale bar, 80 μm. Results of Western blot analysis involving dystrophin antibodies dys1, Mandys18, and Manex46e (recognizing respectively exons 26–30, 17–35, and 46), of total protein extracts (20 μg) obtained from post‐treatment biopsy specimens of Pt 01 (tibialis anterior muscle); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. No bands corresponding to full‐length dystrophin were observed. A schematic representation of the deleted portion of the dystrophin and the region recognized by the used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys18 and Manex46e, of total protein extracts (10–20 μg) obtained from pre‐treatment (performed at time of diagnosis) and post‐treatment biopsy specimens of Pt 02 (tibialis anterior muscle); Ct was used as a positive dystrophin control, CtDmd was used as a dystrophin‐negative control. Below, bands corresponding to myosin heavy chain are shown as a loading control. One faint band corresponding to full‐length dystrophin is observed only in the post‐treatment samples with both Mandys18 and Manex46e antibodies. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys106 (recognizing exon 43) and dys1, of total protein extracts (80 μg, following protein concentration by Amicon Ultra‐0.5 centrifugal filter devices; Millipore) obtained from pre‐treatment and post‐treatment biopsy specimens of Pt 05 (gastrocnemius); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. Bands corresponding approximately to full‐length dystrophin are observed in pre‐ and post‐treatment samples with both antibodies. However, bands in post‐treatment sample appeared higher in amount. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by the used antibodies is depicted above the Western blot. Results of Western blot analysis, involving dystrophin antibodies Mandys106 and dys1, of total protein extracts (80 μg, following protein concentration by Amicon Ultra‐0.5 centrifugal filter devices, Millipore) obtained from pre‐treatment and post‐treatment biopsy specimens of Pt 06 (gastrocnemius); Ct was used as a positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as a loading control. Bands corresponding approximately to full‐length dystrophin are observed in pre‐ and post‐treatment samples with both antibodies. Bands in pre‐treatment sample appeared higher in amount as compared to post‐treatment sample. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by the used antibodies is depicted above the Western blot. Source data are available online for this figure.

    Article Snippet: Pt 05 and Pt 06 (point mutations) showed bands corresponding to dystrophin in both pre‐ and post‐treatment samples, detected only after protein concentration with Amicon Ultra‐0.5 centrifugal filter devices (Millipore) (Fig G and H).

    Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Negative Control, Protein Concentration, Mutagenesis