bovine serum albumin bsa Millipore Search Results


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  • 99
    Millipore bsa
    Atglistatin transiently inhibits lipolysis and protects from HFD-induced obesity. Six weeks old male C57Bl6J mice were fed a HFD (45 kJ% fat; 22.1 kJ g −1 ) for 50 days. Thereafter, mice were fasted for 7 h and then re-fed a HFD with or without Atglistatin (2 mmol kg −1 diet) for 2 h followed by a second, subsequent fasting period of 8 h. ( a ) Plasma FA levels were determined in the 2 h re-fed and 8 h fasted state ( n =5). ( b ) FA release from gonadal adipose tissue explants of re-fed and ( c ) 8 h-fasted mice ( n =5 per group). ( d , e ) Atglistatin does not inhibit human adipocyte lipolysis. ( d ) TG hydrolase activity was assessed in COS-7 lysates of cells overexpressing human and murine ATGL and CGI-58, respectively, in the presence and absence of the indicated concentrations of Atglistatin. ( e ) SGBS and 3T3-L1 preadipocytes were differentiated to adipocytes. Then, cells were preincubated with the indicated concentrations of Atglistatin for 2 h. Thereafter, the medium was replaced by <t>DMEM</t> containing 2% <t>BSA,</t> 10 μM Forskolin and the indicated concentrations of Atglistatin for 1 h. The release of FA in the medium was determined and calculated per mg cell protein. ( f – k ) Mice were fed a HFD for 50 days, followed by HFD-feeding in the presence or absence of Atglistatin for another 50 days. ( f ) Body weight, and ( g ) fat and lean mass development ( n =7 per group). Adipose tissue depots, inguinal (i)WAT, gonadal (g)WAT, and interscapular (i)BAT were analysed for their ( h ) weight and ( i ) adipocyte size. ( j , k ) mRNA expression of IL-6 and macrophage markers F4/80 and Cd11c was assessed in gWAT and iBAT of re-fed mice ( n =5 per group). Data represent mean±s.d. Statistical significance was determined by Student's two-tailed t -test. For analysis of multiple measurements, we performed one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; * P
    Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 59878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bovine fibronectin
    MDCK cells form polarised monolayers on the biochip. MDCK cells stably expressing ML-GFP (green) were grown for three days on a <t>fibronectin-coated</t> glass cover slip (A) and on a fibronectin-coated PDMS-based biochip (B). After fixation, cells were permeabilised and stained with DAPI highlighting nuclei (blue) and antibodies recognising the basolateral marker beta-catenin (magenta) and the tight junction marker ZO-1 (red). A white circle indicates the perimeter of the pore in (B). Displayed are representative x – y sections and x – z sections from the acquired confocal image stack. The black arrowheads indicate the positions of the respective image sections.
    Bovine Fibronectin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bovine ubiquitin
    In vitro experiment demonstrating that AtCHIP is an E3 ligase. Ub, <t>Ubiquitin;</t> E1, ubiquitin-activating enzyme; E2, ubiquitinconjugating enzyme.
    Bovine Ubiquitin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bovine serum fbs
    Colony-forming assay to assess cytotoxicity of 6-subsituted pyrrolo[2,3-d]pyrimidine analogues. IGROV1 EOC (~10 000) cells were plated in 100 mm dishes in complete folate-free <t>RPMI</t> 1640 (pH 7.2), 10% dialyzed <t>FBS,</t> and 25 nM LCV. After 24 h, cells were treated with PMX, 1 , or 11 at varying concentrations (0.1 to 20 μ M) in complete folate-free RPMI 1640 adjusted to pH 6.8 and supplemented with 25 nM LCV. After an additional 24 h, cells were rinsed with PBS, then incubated in complete folate-free RPMI 1640 supplemented with 25 nM LCV (pH 7.2–7.4) for 10 days. Colonies were stained with methylene blue; colony numbers were electronically counted, and results were normalized to the controls. Plots show mean ± SE values, representative of triplicate experiments. IC 50 values ( μ M) were as follows: PMX, > 20 μM; 11, 0.27 μ M, 1, 0.20 μ M.
    Bovine Serum Fbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 556 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore synthetic melanin
    Colony-forming assay to assess cytotoxicity of 6-subsituted pyrrolo[2,3-d]pyrimidine analogues. IGROV1 EOC (~10 000) cells were plated in 100 mm dishes in complete folate-free <t>RPMI</t> 1640 (pH 7.2), 10% dialyzed <t>FBS,</t> and 25 nM LCV. After 24 h, cells were treated with PMX, 1 , or 11 at varying concentrations (0.1 to 20 μ M) in complete folate-free RPMI 1640 adjusted to pH 6.8 and supplemented with 25 nM LCV. After an additional 24 h, cells were rinsed with PBS, then incubated in complete folate-free RPMI 1640 supplemented with 25 nM LCV (pH 7.2–7.4) for 10 days. Colonies were stained with methylene blue; colony numbers were electronically counted, and results were normalized to the controls. Plots show mean ± SE values, representative of triplicate experiments. IC 50 values ( μ M) were as follows: PMX, > 20 μM; 11, 0.27 μ M, 1, 0.20 μ M.
    Synthetic Melanin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 421 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bovine fetuin
    TDA Compliance. Score distributions of the target and decoy N-glycopeptide matches from the mixture of bovine <t>fetuin,</t> <t>lactoferrin,</t> and kappa casein. Data includes both 20 and 40% ACN SPE fractions. a . Without self-consistency. b . With self-consistency.
    Bovine Fetuin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bovine serum albumin bsa fraction v
    TDA Compliance. Score distributions of the target and decoy N-glycopeptide matches from the mixture of bovine <t>fetuin,</t> <t>lactoferrin,</t> and kappa casein. Data includes both 20 and 40% ACN SPE fractions. a . Without self-consistency. b . With self-consistency.
    Bovine Serum Albumin Bsa Fraction V, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 472 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dnase i
    Parachlamydiaceae -induced cell death in S2 cells is accompanied by apoptosis-like morphological and nuclear changes and DNA fragmentation. Panel (A) illustrates morphological and nuclear changes that were observed in S2 cells in response to infection with Parachlamydiaceae , but not Simkania . Insect cells were infected with S. negevensis , Pa. acanthamoebae , or P. amoebophila (MOI 2.5). At 7 h p.i. DNA was stained with DAPI (blue). Untreated cells and cells treated with the apoptosis inducer ActD (7 h) are shown for comparison. The bar corresponds to 10 µm. In (B) the detection of DNA fragmentation by TUNEL staining in S2 cells infected with Parachlamydiaceae is shown. S2 cells were either left untreated, incubated with ActD (10 h), or infected with Pa. acanthamoebae or P. amoebophila (MOI 2.5, 10 h). Bacteria were detected by immunostaining using antibodies raised against purified bacteria (red). TUNEL-positive nuclei are shown in green. Two additional controls were included, a negative control where TdT was omitted from the TUNEL reaction mixture and a positive control where cells were preincubated with <t>DNase</t> I to experimentally introduce DNA double strand breaks in all (also non-apoptotic) cells. Note that apart from this control, TUNEL-positive cells typically display other characteristic features of apoptotic cells, such as condensed and fragmented nuclei and formation of apoptotic bodies. After infection, TUNEL-positive cells were also frequently associated with bacteria. The bar indicates 10 µm.
    Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore facs buffer
    Effect of HPβCD treatment on Npc1 −/− microglia. ( A ) Representative <t>FACS</t> analysis of 7-week-old Npc1 +/+ and Npc1 −/− mice injected twice a week with either phosphate buffered saline <t>(PBS)</t> or HPβCD starting at day of life 7. Cell size (FSC-A) and granularity (SSC-A) returned toward normal in the HPβCD compared to PBS treated mutant mice. ( B ) IBA1 staining (green) in the cerebellum, thalamus and frontal cortex of 7-week-old Npc1 +/+ and Npc1 −/− mice treated with either PBS or HPβCD is consistent with a less activated morphology in the HPβCD treated mice. Scale bar: 20 µm. ( C ) IBA1 (green), CD68 (red) double staining in the thalamus tissue from 7-week-old Npc1 +/+ and Npc1 −/− mice treated with either PBS or HPβCD demonstrates decreased microglial activation in HPβCD treated Npc1 −/− mice. Scale bar: 20 µm. ( D ) FACS analysis of CD11b, CX 3 CR1, unesterified cholesterol and GM1 levels in microglia from Npc1 +/+ treated with PBS (red line) and from Npc1 −/− treated with either PBS (light blue shading) or HPβCD (dark blue line). ( E ) Total ROS production, ascertained by DCFDA staining of isolated microglia from 7-week old control and mutant mice, confirms decreased ROS in HPβCD treated mutant mice. Results are from three independent experiments with ≥3 samples per experiment. Each sample consisted of 10 000 microglial cells. ( F ) Microglial gene expression changes comparing Npc1 −/− mice treated with PBS (dark blue) or HPβCD (light blue) versus Npc1 +/+ injected with PBS and HPβCD. The 207 genes with padj
    Facs Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Atglistatin transiently inhibits lipolysis and protects from HFD-induced obesity. Six weeks old male C57Bl6J mice were fed a HFD (45 kJ% fat; 22.1 kJ g −1 ) for 50 days. Thereafter, mice were fasted for 7 h and then re-fed a HFD with or without Atglistatin (2 mmol kg −1 diet) for 2 h followed by a second, subsequent fasting period of 8 h. ( a ) Plasma FA levels were determined in the 2 h re-fed and 8 h fasted state ( n =5). ( b ) FA release from gonadal adipose tissue explants of re-fed and ( c ) 8 h-fasted mice ( n =5 per group). ( d , e ) Atglistatin does not inhibit human adipocyte lipolysis. ( d ) TG hydrolase activity was assessed in COS-7 lysates of cells overexpressing human and murine ATGL and CGI-58, respectively, in the presence and absence of the indicated concentrations of Atglistatin. ( e ) SGBS and 3T3-L1 preadipocytes were differentiated to adipocytes. Then, cells were preincubated with the indicated concentrations of Atglistatin for 2 h. Thereafter, the medium was replaced by DMEM containing 2% BSA, 10 μM Forskolin and the indicated concentrations of Atglistatin for 1 h. The release of FA in the medium was determined and calculated per mg cell protein. ( f – k ) Mice were fed a HFD for 50 days, followed by HFD-feeding in the presence or absence of Atglistatin for another 50 days. ( f ) Body weight, and ( g ) fat and lean mass development ( n =7 per group). Adipose tissue depots, inguinal (i)WAT, gonadal (g)WAT, and interscapular (i)BAT were analysed for their ( h ) weight and ( i ) adipocyte size. ( j , k ) mRNA expression of IL-6 and macrophage markers F4/80 and Cd11c was assessed in gWAT and iBAT of re-fed mice ( n =5 per group). Data represent mean±s.d. Statistical significance was determined by Student's two-tailed t -test. For analysis of multiple measurements, we performed one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; * P

    Journal: Nature Communications

    Article Title: Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice

    doi: 10.1038/ncomms14859

    Figure Lengend Snippet: Atglistatin transiently inhibits lipolysis and protects from HFD-induced obesity. Six weeks old male C57Bl6J mice were fed a HFD (45 kJ% fat; 22.1 kJ g −1 ) for 50 days. Thereafter, mice were fasted for 7 h and then re-fed a HFD with or without Atglistatin (2 mmol kg −1 diet) for 2 h followed by a second, subsequent fasting period of 8 h. ( a ) Plasma FA levels were determined in the 2 h re-fed and 8 h fasted state ( n =5). ( b ) FA release from gonadal adipose tissue explants of re-fed and ( c ) 8 h-fasted mice ( n =5 per group). ( d , e ) Atglistatin does not inhibit human adipocyte lipolysis. ( d ) TG hydrolase activity was assessed in COS-7 lysates of cells overexpressing human and murine ATGL and CGI-58, respectively, in the presence and absence of the indicated concentrations of Atglistatin. ( e ) SGBS and 3T3-L1 preadipocytes were differentiated to adipocytes. Then, cells were preincubated with the indicated concentrations of Atglistatin for 2 h. Thereafter, the medium was replaced by DMEM containing 2% BSA, 10 μM Forskolin and the indicated concentrations of Atglistatin for 1 h. The release of FA in the medium was determined and calculated per mg cell protein. ( f – k ) Mice were fed a HFD for 50 days, followed by HFD-feeding in the presence or absence of Atglistatin for another 50 days. ( f ) Body weight, and ( g ) fat and lean mass development ( n =7 per group). Adipose tissue depots, inguinal (i)WAT, gonadal (g)WAT, and interscapular (i)BAT were analysed for their ( h ) weight and ( i ) adipocyte size. ( j , k ) mRNA expression of IL-6 and macrophage markers F4/80 and Cd11c was assessed in gWAT and iBAT of re-fed mice ( n =5 per group). Data represent mean±s.d. Statistical significance was determined by Student's two-tailed t -test. For analysis of multiple measurements, we performed one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; * P

    Article Snippet: In brief, cells were preincubated with different concentrations of Atglistatin for 2 h. Then, the medium was replaced by DMEM containing 2% BSA (FA free, Sigma), 10 μM Forskolin and different concentrations of Atglistatin for 1 h. The release of FA in the medium was determined using commercial kits (NEFA C, WAKO).

    Techniques: Mouse Assay, Activity Assay, Expressing, Two Tailed Test

    Dexamethasone suppresses AA-induced cytokine release in COPD and non-COPD. Pulmonary fibroblasts from COPD ( n = 3) and non-COPD ( n = 6) patients were pre-treated with dexamethasone (0.001 - 1 μM) for 60 min prior to challenge with AA (100 μM) in 0.1% BSA-DMEM for 48 h. Cell free supernatants were collected and CXCL8 ( a ) and IL-6 ( b ) release was measured using ELISA. All data are expressed as % of AA-induced cytokine release ± standard error of the mean. Two-way ANOVA with Bonferroni post-hoc testing was used to determine statistical significance. There were no statistical differences

    Journal: Respiratory Research

    Article Title: Dietary ω-6 polyunsaturated fatty acid arachidonic acid increases inflammation, but inhibits ECM protein expression in COPD

    doi: 10.1186/s12931-018-0919-4

    Figure Lengend Snippet: Dexamethasone suppresses AA-induced cytokine release in COPD and non-COPD. Pulmonary fibroblasts from COPD ( n = 3) and non-COPD ( n = 6) patients were pre-treated with dexamethasone (0.001 - 1 μM) for 60 min prior to challenge with AA (100 μM) in 0.1% BSA-DMEM for 48 h. Cell free supernatants were collected and CXCL8 ( a ) and IL-6 ( b ) release was measured using ELISA. All data are expressed as % of AA-induced cytokine release ± standard error of the mean. Two-way ANOVA with Bonferroni post-hoc testing was used to determine statistical significance. There were no statistical differences

    Article Snippet: When the cells reached 80% confluency, they were serum starved by incubation in DMEM (Gibco, Grand Island, New York, US) supplemented with 0.1% bovine serum albumin (BSA) (Sigma Aldrich, Castle Hill, NSW, Australia) and 1% antibiotic-antimycotic for 24 h prior to stimulation.

    Techniques: Enzyme-linked Immunosorbent Assay

    Reduced basal fibronectin, type I collagen, tenascin and perlecan deposition upon arachidonic acid challenge. Pulmonary fibroblasts from COPD ( n = 5–6 ) and non-COPD patients ( n = 4–5 ) were unstimulated (control) or challenged with the ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 72 h. Deposition of fibronectin ( a ), type I collagen ( b ), tenascin ( c ) and perlecan ( d ) into the extracellular matrix (ECM) was measured by ECM ELISA. All data are expressed at fold change compared to control ± standard error of the mean. Challenge with AA is compared to control using a Two-way ANOVA with LSD fisher’s test. Significance is represented as * ( p

    Journal: Respiratory Research

    Article Title: Dietary ω-6 polyunsaturated fatty acid arachidonic acid increases inflammation, but inhibits ECM protein expression in COPD

    doi: 10.1186/s12931-018-0919-4

    Figure Lengend Snippet: Reduced basal fibronectin, type I collagen, tenascin and perlecan deposition upon arachidonic acid challenge. Pulmonary fibroblasts from COPD ( n = 5–6 ) and non-COPD patients ( n = 4–5 ) were unstimulated (control) or challenged with the ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 72 h. Deposition of fibronectin ( a ), type I collagen ( b ), tenascin ( c ) and perlecan ( d ) into the extracellular matrix (ECM) was measured by ECM ELISA. All data are expressed at fold change compared to control ± standard error of the mean. Challenge with AA is compared to control using a Two-way ANOVA with LSD fisher’s test. Significance is represented as * ( p

    Article Snippet: When the cells reached 80% confluency, they were serum starved by incubation in DMEM (Gibco, Grand Island, New York, US) supplemented with 0.1% bovine serum albumin (BSA) (Sigma Aldrich, Castle Hill, NSW, Australia) and 1% antibiotic-antimycotic for 24 h prior to stimulation.

    Techniques: Enzyme-linked Immunosorbent Assay

    Reduced basal fibronectin and type I collagen expression upon arachidonic acid challenge. Pulmonary fibroblasts from COPD patients ( n = 5 ) and non-COPD ( n = 3–4 ) were unstimulated (control) or challenged with the ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (10 and 100 μM) for 48 h. Total RNA was collected and fibronectin ( a ), Type I collagen (1A2) ( b ) and tenascin ( c ) were detected using real time PCR array. The results are normalized to the endogenous control (18S rRNA), and presented as fold change from control ± standard error of the mean. Challenge with AA is compared to control using a Two-way ANOVA with LSD fisher’s test. Significance is represented as * ( p

    Journal: Respiratory Research

    Article Title: Dietary ω-6 polyunsaturated fatty acid arachidonic acid increases inflammation, but inhibits ECM protein expression in COPD

    doi: 10.1186/s12931-018-0919-4

    Figure Lengend Snippet: Reduced basal fibronectin and type I collagen expression upon arachidonic acid challenge. Pulmonary fibroblasts from COPD patients ( n = 5 ) and non-COPD ( n = 3–4 ) were unstimulated (control) or challenged with the ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (10 and 100 μM) for 48 h. Total RNA was collected and fibronectin ( a ), Type I collagen (1A2) ( b ) and tenascin ( c ) were detected using real time PCR array. The results are normalized to the endogenous control (18S rRNA), and presented as fold change from control ± standard error of the mean. Challenge with AA is compared to control using a Two-way ANOVA with LSD fisher’s test. Significance is represented as * ( p

    Article Snippet: When the cells reached 80% confluency, they were serum starved by incubation in DMEM (Gibco, Grand Island, New York, US) supplemented with 0.1% bovine serum albumin (BSA) (Sigma Aldrich, Castle Hill, NSW, Australia) and 1% antibiotic-antimycotic for 24 h prior to stimulation.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Reduced cytokine release upon arachidonic acid challenge in COPD versus non-COPD. Pulmonary fibroblasts from COPD ( n = 11–19) and non-COPD patients ( n = 12–36 ) were unstimulated (control) or challenged with ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (10 and 100 μM) for 48 h. Cell free supernatants were collected and CXCL8 ( a ), IL-6 ( b ) and PGE2 ( c ) release was measured using ELISA or enzyme immunoassay. Other cells from COPD ( n = 6–7) and non-COPD ( n = 6–7) patients were pre-treated with indomethacin (1 μM) ( d, e ) or celecoxib (0.01-1 μM) ( f, g ) for 60 min prior to challenge with AA (100 μM) in 0.1% BSA-DMEM for 48 h. Cell free supernatants were collected and CXCL8 ( d, f ) and IL-6 ( e, g ) release was measured using ELISA. Data are expressed as mean ± standard error of the mean ( a-c ) or as % of AA-induced cytokine release ± standard error of the mean ( d-g ). Two-way ANOVA with Bonferroni post-hoc testing was used to determine statistical significance. Significance is represented as *** ( p

    Journal: Respiratory Research

    Article Title: Dietary ω-6 polyunsaturated fatty acid arachidonic acid increases inflammation, but inhibits ECM protein expression in COPD

    doi: 10.1186/s12931-018-0919-4

    Figure Lengend Snippet: Reduced cytokine release upon arachidonic acid challenge in COPD versus non-COPD. Pulmonary fibroblasts from COPD ( n = 11–19) and non-COPD patients ( n = 12–36 ) were unstimulated (control) or challenged with ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (10 and 100 μM) for 48 h. Cell free supernatants were collected and CXCL8 ( a ), IL-6 ( b ) and PGE2 ( c ) release was measured using ELISA or enzyme immunoassay. Other cells from COPD ( n = 6–7) and non-COPD ( n = 6–7) patients were pre-treated with indomethacin (1 μM) ( d, e ) or celecoxib (0.01-1 μM) ( f, g ) for 60 min prior to challenge with AA (100 μM) in 0.1% BSA-DMEM for 48 h. Cell free supernatants were collected and CXCL8 ( d, f ) and IL-6 ( e, g ) release was measured using ELISA. Data are expressed as mean ± standard error of the mean ( a-c ) or as % of AA-induced cytokine release ± standard error of the mean ( d-g ). Two-way ANOVA with Bonferroni post-hoc testing was used to determine statistical significance. Significance is represented as *** ( p

    Article Snippet: When the cells reached 80% confluency, they were serum starved by incubation in DMEM (Gibco, Grand Island, New York, US) supplemented with 0.1% bovine serum albumin (BSA) (Sigma Aldrich, Castle Hill, NSW, Australia) and 1% antibiotic-antimycotic for 24 h prior to stimulation.

    Techniques: Enzyme-linked Immunosorbent Assay

    Similar arachidonic acid-induced COX-2 mRNA expression in COPD and non-COPD. Pulmonary fibroblasts from COPD ( n = 5) and non-COPD patients ( n = 5 ) were unstimulated (control) or challenged with ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 6 ( a ), 9 ( b ), 24 ( c ) or 48 ( d ) hours. Total RNA was extracted and cyclooxygenase (COX)-2 mRNA expression was measured using qPCR. The results are normalized to the endogenous control (18S rRNA), and presented as fold change from control ( t = 0 h) ± standard error of the mean. Unpaired t-test was used to determined statistical significance. There were no statistical differences

    Journal: Respiratory Research

    Article Title: Dietary ω-6 polyunsaturated fatty acid arachidonic acid increases inflammation, but inhibits ECM protein expression in COPD

    doi: 10.1186/s12931-018-0919-4

    Figure Lengend Snippet: Similar arachidonic acid-induced COX-2 mRNA expression in COPD and non-COPD. Pulmonary fibroblasts from COPD ( n = 5) and non-COPD patients ( n = 5 ) were unstimulated (control) or challenged with ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 6 ( a ), 9 ( b ), 24 ( c ) or 48 ( d ) hours. Total RNA was extracted and cyclooxygenase (COX)-2 mRNA expression was measured using qPCR. The results are normalized to the endogenous control (18S rRNA), and presented as fold change from control ( t = 0 h) ± standard error of the mean. Unpaired t-test was used to determined statistical significance. There were no statistical differences

    Article Snippet: When the cells reached 80% confluency, they were serum starved by incubation in DMEM (Gibco, Grand Island, New York, US) supplemented with 0.1% bovine serum albumin (BSA) (Sigma Aldrich, Castle Hill, NSW, Australia) and 1% antibiotic-antimycotic for 24 h prior to stimulation.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Similar cytokine release upon TNFα challenge in COPD versus non-COPD. Pulmonary fibroblasts from COPD ( n = 15 ) and non-COPD patients ( n = 27 ) were unstimulated (control) or challenged with TNFα in 0.1% BSA-DMEM (1 ng/ml) for 48 h. Cell free supernatants were collected and CXCL8 ( a ) and IL-6 ( b ) release was measured using ELISA. All data are represented as mean ± standard error of the mean. Two-way ANOVA with Bonferroni post-hoc testing was used to determine statistical significance. There were no statistical differences

    Journal: Respiratory Research

    Article Title: Dietary ω-6 polyunsaturated fatty acid arachidonic acid increases inflammation, but inhibits ECM protein expression in COPD

    doi: 10.1186/s12931-018-0919-4

    Figure Lengend Snippet: Similar cytokine release upon TNFα challenge in COPD versus non-COPD. Pulmonary fibroblasts from COPD ( n = 15 ) and non-COPD patients ( n = 27 ) were unstimulated (control) or challenged with TNFα in 0.1% BSA-DMEM (1 ng/ml) for 48 h. Cell free supernatants were collected and CXCL8 ( a ) and IL-6 ( b ) release was measured using ELISA. All data are represented as mean ± standard error of the mean. Two-way ANOVA with Bonferroni post-hoc testing was used to determine statistical significance. There were no statistical differences

    Article Snippet: When the cells reached 80% confluency, they were serum starved by incubation in DMEM (Gibco, Grand Island, New York, US) supplemented with 0.1% bovine serum albumin (BSA) (Sigma Aldrich, Castle Hill, NSW, Australia) and 1% antibiotic-antimycotic for 24 h prior to stimulation.

    Techniques: Enzyme-linked Immunosorbent Assay

    Arachidonic acid does not cause cytotoxicity in COPD and non-COPD fibroblasts. Pulmonary fibroblasts from COPD ( n = 7) and non-COPD patients ( n = 9 ) were unstimulated (control) or challenged with ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 48 h. Cell free supernatants were collected and cell viability was estimated using lactate dehydrogenase (LDH) activity assay. Data is expressed as the absorbance (OD) at 490 nm ± standard error of the mean. Two-way ANOVA with Bonferroni post-hoc testing was used to determine statistical significance. There were no statistical differences

    Journal: Respiratory Research

    Article Title: Dietary ω-6 polyunsaturated fatty acid arachidonic acid increases inflammation, but inhibits ECM protein expression in COPD

    doi: 10.1186/s12931-018-0919-4

    Figure Lengend Snippet: Arachidonic acid does not cause cytotoxicity in COPD and non-COPD fibroblasts. Pulmonary fibroblasts from COPD ( n = 7) and non-COPD patients ( n = 9 ) were unstimulated (control) or challenged with ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 48 h. Cell free supernatants were collected and cell viability was estimated using lactate dehydrogenase (LDH) activity assay. Data is expressed as the absorbance (OD) at 490 nm ± standard error of the mean. Two-way ANOVA with Bonferroni post-hoc testing was used to determine statistical significance. There were no statistical differences

    Article Snippet: When the cells reached 80% confluency, they were serum starved by incubation in DMEM (Gibco, Grand Island, New York, US) supplemented with 0.1% bovine serum albumin (BSA) (Sigma Aldrich, Castle Hill, NSW, Australia) and 1% antibiotic-antimycotic for 24 h prior to stimulation.

    Techniques: Activity Assay

    Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with PBS containing 0.5% BSA and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).

    Journal: PLoS ONE

    Article Title: Involvement of Band3 in the efflux of sphingosine 1-phosphate from erythrocytes

    doi: 10.1371/journal.pone.0177543

    Figure Lengend Snippet: Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with PBS containing 0.5% BSA and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).

    Article Snippet: Then, we replaced the supernatant with PBS containing 0.5% fatty acid-free BSA (A8806; Sigma-Aldrich Co.) and incubated the cells at 37°C for another 20 minutes.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Tube Formation Assay, Incubation, Activity Assay, Polymerase Chain Reaction, Western Blot, Positive Control

    Localisation of the hBD2 peptide in epithelial bronchial 16HBE cells . 16HBE cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with PBS-BSA, the cells were exposed to either latex beads, ethanol fixed conidia or ethanol fixed HF for 18 hours. Il-1β was used as a positive control. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS-Triton 0.05%, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, A. fumigatus fixed organisms and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: Localisation of the hBD2 peptide in epithelial bronchial 16HBE cells . 16HBE cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with PBS-BSA, the cells were exposed to either latex beads, ethanol fixed conidia or ethanol fixed HF for 18 hours. Il-1β was used as a positive control. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS-Triton 0.05%, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, A. fumigatus fixed organisms and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Article Snippet: After washing the cover slips with 5% BSA/PBS (BSA, Fraction V, Sigma), the cells were exposed to either 106 fixed conidia or to 20 μl of the fixed HF solution (20 mg of dry weight/ml), or 5 × 106 latex beads for 24 hours.

    Techniques: Positive Control, Incubation, Fluorescence, Microscopy, Staining

    Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Article Snippet: After washing the cover slips with 5% BSA/PBS (BSA, Fraction V, Sigma), the cells were exposed to either 106 fixed conidia or to 20 μl of the fixed HF solution (20 mg of dry weight/ml), or 5 × 106 latex beads for 24 hours.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Isolation, Amplification, Immunofluorescence, Positive Control, Fluorescence, Microscopy, Staining

    Effects of hyaluronidase on the ability of supernatants from ECs to activate neutrophils morphologically. ECs were cultured for 3 or 20 days and treated with 1 or 100 U/ml TNF for 4 hours. Medium was removed and replaced with PBS/BSA with or without 30 mU/ml hyaluronidase for 30 minutes. The supernatants were collected and added to neutrophils for 5 minutes and their shape change was observed using a microscope to determine the percent activated. For comparison, neutrophils were also treated with PBS/BSA with or without hyaluronidase. Data are mean ± SEM for 3 experiments. ⁎p

    Journal: Experimental Cell Research

    Article Title: A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

    doi: 10.1016/j.yexcr.2009.08.012

    Figure Lengend Snippet: Effects of hyaluronidase on the ability of supernatants from ECs to activate neutrophils morphologically. ECs were cultured for 3 or 20 days and treated with 1 or 100 U/ml TNF for 4 hours. Medium was removed and replaced with PBS/BSA with or without 30 mU/ml hyaluronidase for 30 minutes. The supernatants were collected and added to neutrophils for 5 minutes and their shape change was observed using a microscope to determine the percent activated. For comparison, neutrophils were also treated with PBS/BSA with or without hyaluronidase. Data are mean ± SEM for 3 experiments. ⁎p

    Article Snippet: The monolayer was then treated with 2 ml of PBS/BSA with or without 30 mU/ml heparinase (Oxford Glycosciences, Oxford, UK) or 30 U/ml hyaluronidase (Sigma) for 15 minutes immediately prior to the assay.

    Techniques: Cell Culture, Microscopy

    Effect of heparinase treatment of ECs on (A) adhesion or (B) transmigration of neutrophils. ECs were cultured for 20 days, stimulated with 1 or 100 U/ml TNF for 4 hours, then treated with PBS/BSA with or without 30 mU/ml heparinase for 30 minutes prior to assay. Adhesion and transmigration efficiency are shown for heparinase-treated ECs relative to untreated ECs. Data are mean ± SEM from 3 experiments.

    Journal: Experimental Cell Research

    Article Title: A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

    doi: 10.1016/j.yexcr.2009.08.012

    Figure Lengend Snippet: Effect of heparinase treatment of ECs on (A) adhesion or (B) transmigration of neutrophils. ECs were cultured for 20 days, stimulated with 1 or 100 U/ml TNF for 4 hours, then treated with PBS/BSA with or without 30 mU/ml heparinase for 30 minutes prior to assay. Adhesion and transmigration efficiency are shown for heparinase-treated ECs relative to untreated ECs. Data are mean ± SEM from 3 experiments.

    Article Snippet: The monolayer was then treated with 2 ml of PBS/BSA with or without 30 mU/ml heparinase (Oxford Glycosciences, Oxford, UK) or 30 U/ml hyaluronidase (Sigma) for 15 minutes immediately prior to the assay.

    Techniques: Transmigration Assay, Cell Culture

    Effect of hyaluronidase treatment of ECs on (A) adhesion or (B) transmigration of neutrophils. ECs were cultured for 3 or 20 days, stimulated with 1 or 100 U/ml TNF for 4 hours, then treated with PBS/BSA with or without 30 mU/ml hyaluronidase for 30 minutes prior to the assay. Adhesion and transmigration efficiency are shown for heparinase-treated ECs relative to untreated ECs. Data are mean ± SEM from 4 experiments. ⁎⁎p

    Journal: Experimental Cell Research

    Article Title: A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

    doi: 10.1016/j.yexcr.2009.08.012

    Figure Lengend Snippet: Effect of hyaluronidase treatment of ECs on (A) adhesion or (B) transmigration of neutrophils. ECs were cultured for 3 or 20 days, stimulated with 1 or 100 U/ml TNF for 4 hours, then treated with PBS/BSA with or without 30 mU/ml hyaluronidase for 30 minutes prior to the assay. Adhesion and transmigration efficiency are shown for heparinase-treated ECs relative to untreated ECs. Data are mean ± SEM from 4 experiments. ⁎⁎p

    Article Snippet: The monolayer was then treated with 2 ml of PBS/BSA with or without 30 mU/ml heparinase (Oxford Glycosciences, Oxford, UK) or 30 U/ml hyaluronidase (Sigma) for 15 minutes immediately prior to the assay.

    Techniques: Transmigration Assay, Cell Culture

    Dispersion profiles of ( a ) LYZ and ( b ) BSA at different concentrations. For direct visual comparison of R 1ρ ( triangles ) and R 1 ( circles ), R 1ρ data were multiplied by 10/3 (see ESM, Eqs. S1–S5). R 2 ’s ( squares ) were measured at 20 MHz and are shown in a separate column of each plot. Solid lines provide the best fit result. Uncertain data points (BSA at ω 0 /2π

    Journal: Journal of Biomolecular Nmr

    Article Title: The “long tail” of the protein tumbling correlation function: observation by 1H NMR relaxometry in a wide frequency and concentration range

    doi: 10.1007/s10858-015-0001-1

    Figure Lengend Snippet: Dispersion profiles of ( a ) LYZ and ( b ) BSA at different concentrations. For direct visual comparison of R 1ρ ( triangles ) and R 1 ( circles ), R 1ρ data were multiplied by 10/3 (see ESM, Eqs. S1–S5). R 2 ’s ( squares ) were measured at 20 MHz and are shown in a separate column of each plot. Solid lines provide the best fit result. Uncertain data points (BSA at ω 0 /2π

    Article Snippet: Sample preparation Lysozyme (LYZ), MW = 14,300, from chicken egg white and fatty acid free bovine serum albumin (BSA), MW = 66,500, were delivered from Sigma-Aldrich (product numbers 62970 and A7030, respectively) as lyophilized powders.

    Techniques:

    Order parameter \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ S_{\text{rot}}^{2} $$\end{document} S rot 2 and correlation time of the slow component \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \tau_{\text{S}} $$\end{document} τ S (T = 20 °C) of rotational diffusion as a function of protein concentration of LYZ ( circles ) and BSA ( squares and diamonds ). For BSA, two sets of data are shown corresponding to the analyses using the simple and the more complicated form of the correlation function that includes oligomers (see ESM, Table S2). At the lowest concentration (65 g/L), the upper and lower boundaries of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ S_{\text{rot}}^{2} $$\end{document} S rot 2 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \tau_{\text{S}} , $$\end{document} τ S , respectively, are shown (indicated by arrows ). The minimum (65 g/L) and maximum (260 g/L) concentrations correspond to 4.5 and 18.2 mM for LYZ and 1 and 3.9 mM for BSA, respectively

    Journal: Journal of Biomolecular Nmr

    Article Title: The “long tail” of the protein tumbling correlation function: observation by 1H NMR relaxometry in a wide frequency and concentration range

    doi: 10.1007/s10858-015-0001-1

    Figure Lengend Snippet: Order parameter \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ S_{\text{rot}}^{2} $$\end{document} S rot 2 and correlation time of the slow component \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \tau_{\text{S}} $$\end{document} τ S (T = 20 °C) of rotational diffusion as a function of protein concentration of LYZ ( circles ) and BSA ( squares and diamonds ). For BSA, two sets of data are shown corresponding to the analyses using the simple and the more complicated form of the correlation function that includes oligomers (see ESM, Table S2). At the lowest concentration (65 g/L), the upper and lower boundaries of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ S_{\text{rot}}^{2} $$\end{document} S rot 2 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \tau_{\text{S}} , $$\end{document} τ S , respectively, are shown (indicated by arrows ). The minimum (65 g/L) and maximum (260 g/L) concentrations correspond to 4.5 and 18.2 mM for LYZ and 1 and 3.9 mM for BSA, respectively

    Article Snippet: Sample preparation Lysozyme (LYZ), MW = 14,300, from chicken egg white and fatty acid free bovine serum albumin (BSA), MW = 66,500, were delivered from Sigma-Aldrich (product numbers 62970 and A7030, respectively) as lyophilized powders.

    Techniques: Diffusion-based Assay, Protein Concentration, Concentration Assay

    Size-exclusion chromatogram of LYZ and BSA ( top ) and blue native (BN) PAGE of BSA performed on the elution volume of different peaks of the size-exclusion chromatography ( bottom )

    Journal: Journal of Biomolecular Nmr

    Article Title: The “long tail” of the protein tumbling correlation function: observation by 1H NMR relaxometry in a wide frequency and concentration range

    doi: 10.1007/s10858-015-0001-1

    Figure Lengend Snippet: Size-exclusion chromatogram of LYZ and BSA ( top ) and blue native (BN) PAGE of BSA performed on the elution volume of different peaks of the size-exclusion chromatography ( bottom )

    Article Snippet: Sample preparation Lysozyme (LYZ), MW = 14,300, from chicken egg white and fatty acid free bovine serum albumin (BSA), MW = 66,500, were delivered from Sigma-Aldrich (product numbers 62970 and A7030, respectively) as lyophilized powders.

    Techniques: Polyacrylamide Gel Electrophoresis, Size-exclusion Chromatography

    Effects of BSA and HSA on cytokine release from human adipose tissue. Adipose tissue was incubated for 2, 4, 6, 8, and 24 h in albumin-free medium (blue) and medium supplemented with 1% HSA (red), 1% BSA Fraction V (purple), or 1% BSA Essentially Fatty Acid Free (black). Concentrations of nine cytokines in the conditioned media were analyzed with a multiplex immunoassay; (A) granulocyte-macrophage colony-stimulating factor (B) interferon-γ (C) IL-1β (D) IL-2 (E) IL-6 (F) IL-8 (G) IL-10 (H) IL-12p70 (I) TNF-α. Insets show detailed plots of cytokine release in medium without albumin and in medium with HSA. Concentrations are ng/mL/g adipose tissue for IL-6 and IL-8 and pg/mL/g adipose tissue for the remaining cytokines. Values mean ± SEM (n = 3). Both media containing BSA markedly induced the release of all nine cytokines vs albumin-free medium or medium containing HSA over 24 h (p

    Journal: BMC Endocrine Disorders

    Article Title: Adiponectin, chemerin, cytokines, and dipeptidyl peptidase 4 are released from human adipose tissue in a depot-dependent manner: an in vitro system including human serum albumin

    doi: 10.1186/1472-6823-14-7

    Figure Lengend Snippet: Effects of BSA and HSA on cytokine release from human adipose tissue. Adipose tissue was incubated for 2, 4, 6, 8, and 24 h in albumin-free medium (blue) and medium supplemented with 1% HSA (red), 1% BSA Fraction V (purple), or 1% BSA Essentially Fatty Acid Free (black). Concentrations of nine cytokines in the conditioned media were analyzed with a multiplex immunoassay; (A) granulocyte-macrophage colony-stimulating factor (B) interferon-γ (C) IL-1β (D) IL-2 (E) IL-6 (F) IL-8 (G) IL-10 (H) IL-12p70 (I) TNF-α. Insets show detailed plots of cytokine release in medium without albumin and in medium with HSA. Concentrations are ng/mL/g adipose tissue for IL-6 and IL-8 and pg/mL/g adipose tissue for the remaining cytokines. Values mean ± SEM (n = 3). Both media containing BSA markedly induced the release of all nine cytokines vs albumin-free medium or medium containing HSA over 24 h (p

    Article Snippet: To evaluate the influence of albumin preparation, exploratory incubations for 2, 4, 6, 8, and 24 h were carried out in medium lacking albumin and in medium supplemented with 1% HSA, 1% BSA Fraction V, or 1% BSA Essentially Fatty Acid Free (Sigma-Aldrich, St Louis, MO).

    Techniques: Incubation, Multiplex Assay

    DC-derived IL-15 controls granuloma formation. (A) Spleen cells were obtained from DTR tg mice 24 h after injection of 100 ng DT or PBS and stained with anti-CD11c–PE. The percentages indicate the proportions of DCs in a DC-enriched fraction; i.e., the cells at the interface after a dense BSA gradient centrifugation (see DC preparation… in Materials and methods). (B) Granuloma formation in the liver of P. acnes –primed DT-injected DTR tg mice. On day 3 after a 0.5-mg heat-killed P. acnes injection, livers were taken from WT and DTR tg mice that had been injected with either PBS or DT on the day before P. acnes injection, and sections were stained with H E. Numbers of granulomas were counted in five different fields under a microscope. Values represent SD ( n = 3 mice/group). Data are representative of three experiments. (C) To test whether DCs were present in the granuloma regions, the liver sections were stained with biotinylated anti-CD11c mAb and streptavidin-HRP and further visualized with DAB. Slides were counterstained with Mayer's hematoxylin. Bar, 100 μm. (D) IL-12p70, IFN-γ, and chemokine levels in the sera of DT-injected DTR tg mice were assessed by ELISA at 72 h after a 0.5-mg heat-killed P. acnes injection. Values represent SD ( n = 3 mice/group). Data are representative of three experiments. (E) Immunofluoresence staining for the identification of IL-15–producing cells. Acetone-fixed frozen tissue sections were incubated with FITC-conjugated anti-CD11c (clone N418) and biotinylated anti–IL-15 antibodies and further developed with streptavidin-PE. Bar, 50 μm. (F) Granuloma formation in the liver of zymosan-primed DT-injected DTR tg mice. As described in B and C, livers were taken from the indicated mice on day 3 after a 1-mg zymosan injection, and sections were stained for CD11c. Slides were counterstained with Mayer's hematoxylin. Bar, 100 μm. (G) Granuloma formation in the liver of BMDC-injected IL-15 −/− mice. IL-15 −/− mice were injected with 1 × 10 6 WT BMDCs or IL-15 −/− BMDCs. After 12 h, mice were injected with 0.5 mg P. acnes , and granuloma formation was analyzed 6 d later as described in B, C, and F. Bar, 100 μm.

    Journal: The Journal of Experimental Medicine

    Article Title: Essential roles of DC-derived IL-15 as a mediator of inflammatory responses in vivo

    doi: 10.1084/jem.20061297

    Figure Lengend Snippet: DC-derived IL-15 controls granuloma formation. (A) Spleen cells were obtained from DTR tg mice 24 h after injection of 100 ng DT or PBS and stained with anti-CD11c–PE. The percentages indicate the proportions of DCs in a DC-enriched fraction; i.e., the cells at the interface after a dense BSA gradient centrifugation (see DC preparation… in Materials and methods). (B) Granuloma formation in the liver of P. acnes –primed DT-injected DTR tg mice. On day 3 after a 0.5-mg heat-killed P. acnes injection, livers were taken from WT and DTR tg mice that had been injected with either PBS or DT on the day before P. acnes injection, and sections were stained with H E. Numbers of granulomas were counted in five different fields under a microscope. Values represent SD ( n = 3 mice/group). Data are representative of three experiments. (C) To test whether DCs were present in the granuloma regions, the liver sections were stained with biotinylated anti-CD11c mAb and streptavidin-HRP and further visualized with DAB. Slides were counterstained with Mayer's hematoxylin. Bar, 100 μm. (D) IL-12p70, IFN-γ, and chemokine levels in the sera of DT-injected DTR tg mice were assessed by ELISA at 72 h after a 0.5-mg heat-killed P. acnes injection. Values represent SD ( n = 3 mice/group). Data are representative of three experiments. (E) Immunofluoresence staining for the identification of IL-15–producing cells. Acetone-fixed frozen tissue sections were incubated with FITC-conjugated anti-CD11c (clone N418) and biotinylated anti–IL-15 antibodies and further developed with streptavidin-PE. Bar, 50 μm. (F) Granuloma formation in the liver of zymosan-primed DT-injected DTR tg mice. As described in B and C, livers were taken from the indicated mice on day 3 after a 1-mg zymosan injection, and sections were stained for CD11c. Slides were counterstained with Mayer's hematoxylin. Bar, 100 μm. (G) Granuloma formation in the liver of BMDC-injected IL-15 −/− mice. IL-15 −/− mice were injected with 1 × 10 6 WT BMDCs or IL-15 −/− BMDCs. After 12 h, mice were injected with 0.5 mg P. acnes , and granuloma formation was analyzed 6 d later as described in B, C, and F. Bar, 100 μm.

    Article Snippet: In brief, collagenase-digested spleen cells were suspended in a 28% BSA solution in 1.08 g/ml PBS, overlaid with 1 ml FCS-free RPMI 1640 medium (Sigma-Aldrich), and centrifuged at 9,500 g for 20 min at 4°C.

    Techniques: Derivative Assay, Mouse Assay, Injection, Staining, Gradient Centrifugation, Microscopy, Enzyme-linked Immunosorbent Assay, Incubation

    Adipose tissue deposition and lipolysis in EPRS A/A and EPRS D/D mice a , Length of mice was measured from head to beginning of tail using a digital caliper (Fisherbrand Traceable). Data shown are mean ± SEM, n = 15 for 20-week male mice. b , Ventral view of wild-type and EPRS A/A mice abdominal cavity. c , Weights of adipose and non-adipose tissues from 20-week male EPRS D/D and control mice (mean ± SEM, n = 14/group, P value from unpaired t-test). d , Scanning electron micrographs of EWAT in 20-week male EPRS S/S , EPRS A/A , and EPRS D/D mice. e , Total adipocyte cell number in EWAT of EPRS A/A knock-in and wild-type mice. Data represent mean ± SEM, n = 5/group. f , Elevated lipolysis in adipocytes from EPRS A/A , but not EPRS D/D , mice (mean ± SEM; n = 6/group). g , Elevated β-oxidation in WAT explants from EPRS A/A mice as determined by release of 14 CO 2 from 14 C-oleic acid (mean ± SEM; n = 5/group). h , Serum levels of insulin, glucose, triglycerides (TG) and free fatty acids (FFA) in 12-h fasted and 1-h post-prandial (fed) 16-week old male mice (mean ± SEM, n = 10/group, * P

    Journal: Nature

    Article Title: EPRS is a critical mTORC1-S6K1 effector that influences adiposity in mice

    doi: 10.1038/nature21380

    Figure Lengend Snippet: Adipose tissue deposition and lipolysis in EPRS A/A and EPRS D/D mice a , Length of mice was measured from head to beginning of tail using a digital caliper (Fisherbrand Traceable). Data shown are mean ± SEM, n = 15 for 20-week male mice. b , Ventral view of wild-type and EPRS A/A mice abdominal cavity. c , Weights of adipose and non-adipose tissues from 20-week male EPRS D/D and control mice (mean ± SEM, n = 14/group, P value from unpaired t-test). d , Scanning electron micrographs of EWAT in 20-week male EPRS S/S , EPRS A/A , and EPRS D/D mice. e , Total adipocyte cell number in EWAT of EPRS A/A knock-in and wild-type mice. Data represent mean ± SEM, n = 5/group. f , Elevated lipolysis in adipocytes from EPRS A/A , but not EPRS D/D , mice (mean ± SEM; n = 6/group). g , Elevated β-oxidation in WAT explants from EPRS A/A mice as determined by release of 14 CO 2 from 14 C-oleic acid (mean ± SEM; n = 5/group). h , Serum levels of insulin, glucose, triglycerides (TG) and free fatty acids (FFA) in 12-h fasted and 1-h post-prandial (fed) 16-week old male mice (mean ± SEM, n = 10/group, * P

    Article Snippet: Briefly, after mouse sacrifice, fat pads were removed and minced in Krebs-Ringer-bicarbonate-HEPES (KRBH) buffer pH 7.4 containing 10 mM sodium bicarbonate, 30 mM HEPES, 200 nM adenosine, and 1% fatty acid-free bovine serum albumin (BSA, Sigma).

    Techniques: Mouse Assay, Knock-In

    CVs showing the regeneration of the electrode surface for subsequent miRNA assays. The regenerated surface was treated with 1% BSA and re-hybridized with 5.0 nM biotin-miRNA, followed by the attachment of Fc-capped gold nanoparticle/streptavidin conjugates.

    Journal: Analytical chemistry

    Article Title: Direct quantification of microRNA at low pM level in sera of glioma patients using a competitive hybridization followed by amplified voltammetric detection

    doi: 10.1021/ac203368h

    Figure Lengend Snippet: CVs showing the regeneration of the electrode surface for subsequent miRNA assays. The regenerated surface was treated with 1% BSA and re-hybridized with 5.0 nM biotin-miRNA, followed by the attachment of Fc-capped gold nanoparticle/streptavidin conjugates.

    Article Snippet: Potassium perchlorate, EDTA, KH PO , K HPO , tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 1-hexanethiol (HT), gold nanoparticle/streptavidin conjugate, 6-ferrocenyl-1-hexanethiol, and bovine serum album (BSA) were acquired from Sigma-Aldrich (St. Louis, MO).

    Techniques:

    Increase in vascular permeability in diabetic athymic nude rats is alleviated with intravitreal ASC injection. (A) Two months post diabetes induction or age matched normal rats were injected intravitreally with saline (n = 8–10/group) or ASC (n = 6–8/group; 250,000 cells in 2 µL of saline). At day 7, rats were injected with 100 mg/kg of FITC-BSA via tail vein. After 1hr, rats were euthanized and perfused with 4% paraformaldehyde, and eyes enucleated and embedded. About ten 10 µm sections of equal area were analyzed for FITC fluorescence and normalized to total plasma fluorescence. A significant (**p

    Journal: PLoS ONE

    Article Title: Regenerative Therapeutic Potential of Adipose Stromal Cells in Early Stage Diabetic Retinopathy

    doi: 10.1371/journal.pone.0084671

    Figure Lengend Snippet: Increase in vascular permeability in diabetic athymic nude rats is alleviated with intravitreal ASC injection. (A) Two months post diabetes induction or age matched normal rats were injected intravitreally with saline (n = 8–10/group) or ASC (n = 6–8/group; 250,000 cells in 2 µL of saline). At day 7, rats were injected with 100 mg/kg of FITC-BSA via tail vein. After 1hr, rats were euthanized and perfused with 4% paraformaldehyde, and eyes enucleated and embedded. About ten 10 µm sections of equal area were analyzed for FITC fluorescence and normalized to total plasma fluorescence. A significant (**p

    Article Snippet: Briefly, athymic nude rats after two months post diabetes induction or age matched normal rats were anesthetized and received tail vein injection of FITC-BSA (100 mg/Kg body weight, Sigma-Aldrich) One hour after injection rats were euthanized, perfused with 4% paraformaldehyde, eyes were enucleated and embedded in Tissue-Tek CRYO-OCT Compound (Thermo Fisher scientific, Inc).

    Techniques: Permeability, Injection, Fluorescence

    Graphical representation of two main designs of glucose and lactate biosensors used in this study: GB1 (Panel A ): Pt c /PPD/[PEI(0.5%)-GOx] 5 /PU (5%); GB2 (Panel B ): Pt c /PPD/[PE(0.5%)-GOx] 5 /GTA(1%)-BSA(2%); LB1 (Panel C ): Pt c /PPD/[PEI(0.5%)-LOx] 5 /PU (5%); LB2 (Panel D ): Pt c /PPD/[PEI(0.5%)-LOx] 5 / GTA(1%)-BSA(2%); Pt c : Pt cylinder 1 mm long, 125 μm diameter; GOx: glucose oxidase; LOx: lactate oxidase PPD: poly-ortho-phenylenediamine; PEI: polyethyleneimine; PU: polyurethane; GTA: glutaraldehyde; BSA: bovine serum albumin. The subscript “x” represents the number of dip−evaporation deposition stages and in brackets the concentration of the component.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Low-Temperature Storage Improves the Over-Time Stability of Implantable Glucose and Lactate Biosensors

    doi: 10.3390/s19020422

    Figure Lengend Snippet: Graphical representation of two main designs of glucose and lactate biosensors used in this study: GB1 (Panel A ): Pt c /PPD/[PEI(0.5%)-GOx] 5 /PU (5%); GB2 (Panel B ): Pt c /PPD/[PE(0.5%)-GOx] 5 /GTA(1%)-BSA(2%); LB1 (Panel C ): Pt c /PPD/[PEI(0.5%)-LOx] 5 /PU (5%); LB2 (Panel D ): Pt c /PPD/[PEI(0.5%)-LOx] 5 / GTA(1%)-BSA(2%); Pt c : Pt cylinder 1 mm long, 125 μm diameter; GOx: glucose oxidase; LOx: lactate oxidase PPD: poly-ortho-phenylenediamine; PEI: polyethyleneimine; PU: polyurethane; GTA: glutaraldehyde; BSA: bovine serum albumin. The subscript “x” represents the number of dip−evaporation deposition stages and in brackets the concentration of the component.

    Article Snippet: Ascorbic acid, D-(+)-glucose, L-lactate, glucose oxidase from Aspergillus Niger (EC 1.1.3.4), lactate oxidase from Pediococcus species (EC 1.1.3.2), albumin from bovine serum (BSA) , o -phenylenediamine (OPD), polyethylenimine (PEI), glutaraldehyde (GTA) , polyurethane (PU) and tetrahydrofuran (THF) were purchased from Sigma-Aldrich (Milano, Italy).

    Techniques: Evaporation, Concentration Assay

    Scattering plot describing the variations of V MAX (Panel A , B ), K M (Panel C , D ) and LRS (Panel E , F ), in a range of 28 days (left inset) and of four months (right inset) of LB1 design Ptc/PPD/[PEI (0.5%)-LOx] 5 / GTA(1%)-BSA (2%) in the defined range of 120 days, when stored at +4 °C (red plot), −20 °C (green plot) and −80 °C (blue plot).

    Journal: Sensors (Basel, Switzerland)

    Article Title: Low-Temperature Storage Improves the Over-Time Stability of Implantable Glucose and Lactate Biosensors

    doi: 10.3390/s19020422

    Figure Lengend Snippet: Scattering plot describing the variations of V MAX (Panel A , B ), K M (Panel C , D ) and LRS (Panel E , F ), in a range of 28 days (left inset) and of four months (right inset) of LB1 design Ptc/PPD/[PEI (0.5%)-LOx] 5 / GTA(1%)-BSA (2%) in the defined range of 120 days, when stored at +4 °C (red plot), −20 °C (green plot) and −80 °C (blue plot).

    Article Snippet: Ascorbic acid, D-(+)-glucose, L-lactate, glucose oxidase from Aspergillus Niger (EC 1.1.3.4), lactate oxidase from Pediococcus species (EC 1.1.3.2), albumin from bovine serum (BSA) , o -phenylenediamine (OPD), polyethylenimine (PEI), glutaraldehyde (GTA) , polyurethane (PU) and tetrahydrofuran (THF) were purchased from Sigma-Aldrich (Milano, Italy).

    Techniques:

    Scattering plot describing the variations of V MAX (Panel A , B ), K M (Panel C , D ) and LRS (Panel E , F ), in a range of 28 days (left inset) and of four months (right inset) of GB2 design Ptc/PPD/[PEI (0.5%)-GOx]5/ GTA(1%)-BSA (2%) when stored at +4 °C (red plot), −20 °C (green plot) and −80 °C (blue plot).

    Journal: Sensors (Basel, Switzerland)

    Article Title: Low-Temperature Storage Improves the Over-Time Stability of Implantable Glucose and Lactate Biosensors

    doi: 10.3390/s19020422

    Figure Lengend Snippet: Scattering plot describing the variations of V MAX (Panel A , B ), K M (Panel C , D ) and LRS (Panel E , F ), in a range of 28 days (left inset) and of four months (right inset) of GB2 design Ptc/PPD/[PEI (0.5%)-GOx]5/ GTA(1%)-BSA (2%) when stored at +4 °C (red plot), −20 °C (green plot) and −80 °C (blue plot).

    Article Snippet: Ascorbic acid, D-(+)-glucose, L-lactate, glucose oxidase from Aspergillus Niger (EC 1.1.3.4), lactate oxidase from Pediococcus species (EC 1.1.3.2), albumin from bovine serum (BSA) , o -phenylenediamine (OPD), polyethylenimine (PEI), glutaraldehyde (GTA) , polyurethane (PU) and tetrahydrofuran (THF) were purchased from Sigma-Aldrich (Milano, Italy).

    Techniques:

    Resistance to IgE-mediated passive cutaneous anaphylaxis in mice with PigA -deficient mast cells. Mice were given injections with IgE anti-DNP in one ear and as control with medium in the other. On the next day, intravenous injections of DNP-BSA mixed

    Journal: Blood

    Article Title: Impaired Fc?RI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins

    doi: 10.1182/blood-2011-02-338053

    Figure Lengend Snippet: Resistance to IgE-mediated passive cutaneous anaphylaxis in mice with PigA -deficient mast cells. Mice were given injections with IgE anti-DNP in one ear and as control with medium in the other. On the next day, intravenous injections of DNP-BSA mixed

    Article Snippet: After stimulation with IgE anti-DNP (IGEL-A2) and DNP-BSA, BMMC were lysed by incubation for 30 minutes on ice in lysis buffer (20mM Tris-HCl, at pH 7.4 containing 0.5% Triton X-100, 150mM NaCl, 2mM EDTA, 50mM NaF, 1mM Na3 VO4 , 1mM PMSF, 10 μg aprotinin/mL, and 10 μg leupeptin/mL).

    Techniques: Mouse Assay

    Impaired IgE/antigen-mediated degranulation of PigA -deficient BMMCs . BMMCs were (A) sensitized with IgE anti-DNP, followed by challenge with various concentrations of DNP-BSA for 1 hour, or (B) activated with the nonspecific degranulation stimulus compound

    Journal: Blood

    Article Title: Impaired Fc?RI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins

    doi: 10.1182/blood-2011-02-338053

    Figure Lengend Snippet: Impaired IgE/antigen-mediated degranulation of PigA -deficient BMMCs . BMMCs were (A) sensitized with IgE anti-DNP, followed by challenge with various concentrations of DNP-BSA for 1 hour, or (B) activated with the nonspecific degranulation stimulus compound

    Article Snippet: After stimulation with IgE anti-DNP (IGEL-A2) and DNP-BSA, BMMC were lysed by incubation for 30 minutes on ice in lysis buffer (20mM Tris-HCl, at pH 7.4 containing 0.5% Triton X-100, 150mM NaCl, 2mM EDTA, 50mM NaF, 1mM Na3 VO4 , 1mM PMSF, 10 μg aprotinin/mL, and 10 μg leupeptin/mL).

    Techniques:

    Normal IgE/antigen-induced FcϵRI aggregation in PigA -deficient BMMCs. BMMCs were sensitized with IgE anti-DNP, and either left in medium (-Ag) or challenged with DNP-BSA antigen (+Ag) for 3 minutes, fixed and stained with fluorescent anti–mouse

    Journal: Blood

    Article Title: Impaired Fc?RI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins

    doi: 10.1182/blood-2011-02-338053

    Figure Lengend Snippet: Normal IgE/antigen-induced FcϵRI aggregation in PigA -deficient BMMCs. BMMCs were sensitized with IgE anti-DNP, and either left in medium (-Ag) or challenged with DNP-BSA antigen (+Ag) for 3 minutes, fixed and stained with fluorescent anti–mouse

    Article Snippet: After stimulation with IgE anti-DNP (IGEL-A2) and DNP-BSA, BMMC were lysed by incubation for 30 minutes on ice in lysis buffer (20mM Tris-HCl, at pH 7.4 containing 0.5% Triton X-100, 150mM NaCl, 2mM EDTA, 50mM NaF, 1mM Na3 VO4 , 1mM PMSF, 10 μg aprotinin/mL, and 10 μg leupeptin/mL).

    Techniques: Staining

    Reduced IgE/antigen-induced FcR γ-chain tyrosine phosphorylation in PigA -deficient BMMCs. After sensitization of BMMCs with IgE anti-DNP for 3 hours at 37°C, DNP-BSA (Ag) was added and the cells were challenged for 3 minutes at 37°C,

    Journal: Blood

    Article Title: Impaired Fc?RI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins

    doi: 10.1182/blood-2011-02-338053

    Figure Lengend Snippet: Reduced IgE/antigen-induced FcR γ-chain tyrosine phosphorylation in PigA -deficient BMMCs. After sensitization of BMMCs with IgE anti-DNP for 3 hours at 37°C, DNP-BSA (Ag) was added and the cells were challenged for 3 minutes at 37°C,

    Article Snippet: After stimulation with IgE anti-DNP (IGEL-A2) and DNP-BSA, BMMC were lysed by incubation for 30 minutes on ice in lysis buffer (20mM Tris-HCl, at pH 7.4 containing 0.5% Triton X-100, 150mM NaCl, 2mM EDTA, 50mM NaF, 1mM Na3 VO4 , 1mM PMSF, 10 μg aprotinin/mL, and 10 μg leupeptin/mL).

    Techniques:

    Effect of nonglycated BSA and AGE-BSA at 40 AU/mL (3.8 mg/mL) on endothelium-dependent vasorelaxation in aortic rings from control ( A ) and MS rats ( B ). Results are expressed as relaxation percentage from the initial precontraction level with NE 1 μM. * P

    Journal: Glycobiology

    Article Title: Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome

    doi: 10.1093/glycob/cwn034

    Figure Lengend Snippet: Effect of nonglycated BSA and AGE-BSA at 40 AU/mL (3.8 mg/mL) on endothelium-dependent vasorelaxation in aortic rings from control ( A ) and MS rats ( B ). Results are expressed as relaxation percentage from the initial precontraction level with NE 1 μM. * P

    Article Snippet: Aliquots of 500 μL containing 10 mg/mL of BSA or AGE-BSA were diluted to 1.5 mL with deionized water and centrifuged in Amicon Ultracel-10 K (Millipore Corporation, MA) at 2,000 × g for 30 min to a final volume of 500 μL.

    Techniques: Mass Spectrometry

    Fluorescence spectrum of AGE-BSA and nonglycated BSA. Both solutions were tested at 2 mg/mL of protein. The peak of fluorescence is not present in the nonglycated BSA. The interrupted line represents the saturation level of the fluorescence detection by the equipment employed.

    Journal: Glycobiology

    Article Title: Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome

    doi: 10.1093/glycob/cwn034

    Figure Lengend Snippet: Fluorescence spectrum of AGE-BSA and nonglycated BSA. Both solutions were tested at 2 mg/mL of protein. The peak of fluorescence is not present in the nonglycated BSA. The interrupted line represents the saturation level of the fluorescence detection by the equipment employed.

    Article Snippet: Aliquots of 500 μL containing 10 mg/mL of BSA or AGE-BSA were diluted to 1.5 mL with deionized water and centrifuged in Amicon Ultracel-10 K (Millipore Corporation, MA) at 2,000 × g for 30 min to a final volume of 500 μL.

    Techniques: Fluorescence

    Purification of AGE-BSA on Affi-Gel-Blue. Three peaks were observed: the first one corresponds to highly glycated BSA which did not bind to the matrix, the second one corresponds to the less glycated BSA which bound moderately, and the third peak corresponds to the stronger bound BSA. The first peak was the only one to be further analyzed. The arrows indicate the buffer used at each stage.

    Journal: Glycobiology

    Article Title: Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome

    doi: 10.1093/glycob/cwn034

    Figure Lengend Snippet: Purification of AGE-BSA on Affi-Gel-Blue. Three peaks were observed: the first one corresponds to highly glycated BSA which did not bind to the matrix, the second one corresponds to the less glycated BSA which bound moderately, and the third peak corresponds to the stronger bound BSA. The first peak was the only one to be further analyzed. The arrows indicate the buffer used at each stage.

    Article Snippet: Aliquots of 500 μL containing 10 mg/mL of BSA or AGE-BSA were diluted to 1.5 mL with deionized water and centrifuged in Amicon Ultracel-10 K (Millipore Corporation, MA) at 2,000 × g for 30 min to a final volume of 500 μL.

    Techniques: Purification

    Evaluation of the specific activity (SA) of AGE-BSA. The increase of specific activity obtained by the purification process on Affi-Gel-Blue is observed. Bold circles represent the nonglycated BSA. Bold squares represent the unpurified AGE-BSA and bold triangles represent the purified AGE-BSA. Specific activity increased in approximately 33% by the purification process.

    Journal: Glycobiology

    Article Title: Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome

    doi: 10.1093/glycob/cwn034

    Figure Lengend Snippet: Evaluation of the specific activity (SA) of AGE-BSA. The increase of specific activity obtained by the purification process on Affi-Gel-Blue is observed. Bold circles represent the nonglycated BSA. Bold squares represent the unpurified AGE-BSA and bold triangles represent the purified AGE-BSA. Specific activity increased in approximately 33% by the purification process.

    Article Snippet: Aliquots of 500 μL containing 10 mg/mL of BSA or AGE-BSA were diluted to 1.5 mL with deionized water and centrifuged in Amicon Ultracel-10 K (Millipore Corporation, MA) at 2,000 × g for 30 min to a final volume of 500 μL.

    Techniques: Activity Assay, Purification

    Analysis of BSA and AGE-BSA by mass spectrometry. The mass increase of protein after glycation is observed. The mass of nonglycated BSA was 66,655.58 Da ( A ) and that of AGE-BSA was 74,461.15 Da ( B ). The net increase of mass was 7,805.57 Da, corresponding to the addition of 48 molecules of glucose to each BSA molecule. Inset: change of isoelectric point of BSA after glycation. Nonglycated BSA had a pI of 4.2 while AGE-BSA had a pI of 6.3. Line 2 is the broad range pI standard kit (pH 3–10).

    Journal: Glycobiology

    Article Title: Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome

    doi: 10.1093/glycob/cwn034

    Figure Lengend Snippet: Analysis of BSA and AGE-BSA by mass spectrometry. The mass increase of protein after glycation is observed. The mass of nonglycated BSA was 66,655.58 Da ( A ) and that of AGE-BSA was 74,461.15 Da ( B ). The net increase of mass was 7,805.57 Da, corresponding to the addition of 48 molecules of glucose to each BSA molecule. Inset: change of isoelectric point of BSA after glycation. Nonglycated BSA had a pI of 4.2 while AGE-BSA had a pI of 6.3. Line 2 is the broad range pI standard kit (pH 3–10).

    Article Snippet: Aliquots of 500 μL containing 10 mg/mL of BSA or AGE-BSA were diluted to 1.5 mL with deionized water and centrifuged in Amicon Ultracel-10 K (Millipore Corporation, MA) at 2,000 × g for 30 min to a final volume of 500 μL.

    Techniques: Mass Spectrometry

    Effect of AGE-BSA on vascular contraction in aortic rings from control (solid bars) and MS rats (open bars). The contractions were induced by NE 1 μM and basal tension was normalized to 100% in control and MS rats. Tension values in grams are shown in Table III . Results are the means ± SEM of six independent experiments. * P

    Journal: Glycobiology

    Article Title: Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome

    doi: 10.1093/glycob/cwn034

    Figure Lengend Snippet: Effect of AGE-BSA on vascular contraction in aortic rings from control (solid bars) and MS rats (open bars). The contractions were induced by NE 1 μM and basal tension was normalized to 100% in control and MS rats. Tension values in grams are shown in Table III . Results are the means ± SEM of six independent experiments. * P

    Article Snippet: Aliquots of 500 μL containing 10 mg/mL of BSA or AGE-BSA were diluted to 1.5 mL with deionized water and centrifuged in Amicon Ultracel-10 K (Millipore Corporation, MA) at 2,000 × g for 30 min to a final volume of 500 μL.

    Techniques: Mass Spectrometry

    MDCK cells form polarised monolayers on the biochip. MDCK cells stably expressing ML-GFP (green) were grown for three days on a fibronectin-coated glass cover slip (A) and on a fibronectin-coated PDMS-based biochip (B). After fixation, cells were permeabilised and stained with DAPI highlighting nuclei (blue) and antibodies recognising the basolateral marker beta-catenin (magenta) and the tight junction marker ZO-1 (red). A white circle indicates the perimeter of the pore in (B). Displayed are representative x – y sections and x – z sections from the acquired confocal image stack. The black arrowheads indicate the positions of the respective image sections.

    Journal: Rsc Advances

    Article Title: A microfluidic biochip for locally confined stimulation of cells within an epithelial monolayer microfluidic biochip for locally confined stimulation of cells within an epithelial monolayer †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra11943g

    doi: 10.1039/c7ra11943g

    Figure Lengend Snippet: MDCK cells form polarised monolayers on the biochip. MDCK cells stably expressing ML-GFP (green) were grown for three days on a fibronectin-coated glass cover slip (A) and on a fibronectin-coated PDMS-based biochip (B). After fixation, cells were permeabilised and stained with DAPI highlighting nuclei (blue) and antibodies recognising the basolateral marker beta-catenin (magenta) and the tight junction marker ZO-1 (red). A white circle indicates the perimeter of the pore in (B). Displayed are representative x – y sections and x – z sections from the acquired confocal image stack. The black arrowheads indicate the positions of the respective image sections.

    Article Snippet: To this end, bovine fibronectin (Sigma Aldrich) was diluted in phosphate buffered saline (PBS) to a concentration of 10 μg ml–1 and a drop of the fibronectin solution of sufficient volume to cover the cell culture area (approximately 10 mm diameter) was dispensed at the chip surface and incubated for 1 h at room temperature.

    Techniques: Stable Transfection, Expressing, Staining, Marker

    Design of the microfluidic biochip. On the top left side, the designs of the three PDMS layers from which the chip is made are shown. The light blue areas in layer 1 correspond to channels, whereas the dark blue areas in layer 2 correspond to holes or pores. The green areas in layer 3 correspond to channels, and the star-shaped structures represent the position of access ports for tubing. In the overlay image (middle right) the fibronectin-coated cell culture area is outlined by an orange oval, the magnification (top right) shows how the pores are aligned above the channels and also a schematic cross section is depicted (bottom right). On the bottom a photograph of a completely assembled chip with a supporting glass cover slip is shown.

    Journal: Rsc Advances

    Article Title: A microfluidic biochip for locally confined stimulation of cells within an epithelial monolayer microfluidic biochip for locally confined stimulation of cells within an epithelial monolayer †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra11943g

    doi: 10.1039/c7ra11943g

    Figure Lengend Snippet: Design of the microfluidic biochip. On the top left side, the designs of the three PDMS layers from which the chip is made are shown. The light blue areas in layer 1 correspond to channels, whereas the dark blue areas in layer 2 correspond to holes or pores. The green areas in layer 3 correspond to channels, and the star-shaped structures represent the position of access ports for tubing. In the overlay image (middle right) the fibronectin-coated cell culture area is outlined by an orange oval, the magnification (top right) shows how the pores are aligned above the channels and also a schematic cross section is depicted (bottom right). On the bottom a photograph of a completely assembled chip with a supporting glass cover slip is shown.

    Article Snippet: To this end, bovine fibronectin (Sigma Aldrich) was diluted in phosphate buffered saline (PBS) to a concentration of 10 μg ml–1 and a drop of the fibronectin solution of sufficient volume to cover the cell culture area (approximately 10 mm diameter) was dispensed at the chip surface and incubated for 1 h at room temperature.

    Techniques: Chromatin Immunoprecipitation, Cell Culture

    SHIP2 is required for in vitro lymphangiogenesis in primary HDLEC. ( A ) MTS cell proliferation assay in response to growth factor stimulation for 48 hrs, normalized to 1% FBS of non-targeting (NT) siRNA (N = 3). ( B ) Cell adhesion assay of siRNA-transfected HDLEC onto BSA, collagen and fibronectin and quantification presented as area fraction of whole images (triplicates per experiment; 5 independent experiments). ( C ) Wound scratch cell migration assay of siRNA-transfected HDLEC immediately after injury, at 24 hr and 48 hrs afterward. ( D ) 3D tube formation networks in response to growth factors imaged 24 hrs post plating. ( E ) Annexin V apoptosis assay of siRNA-transfected HDLEC either untreated or following staurosporine treatment, quantification of total AnnexinV-positive cell populations and mean intensity of AnnexinV and propidium iodide of 3 independent experiments. Data presented as means±SEM. * p

    Journal: PLoS ONE

    Article Title: Evidence for SH2 Domain-Containing 5′-Inositol Phosphatase-2 (SHIP2) Contributing to a Lymphatic Dysfunction

    doi: 10.1371/journal.pone.0112548

    Figure Lengend Snippet: SHIP2 is required for in vitro lymphangiogenesis in primary HDLEC. ( A ) MTS cell proliferation assay in response to growth factor stimulation for 48 hrs, normalized to 1% FBS of non-targeting (NT) siRNA (N = 3). ( B ) Cell adhesion assay of siRNA-transfected HDLEC onto BSA, collagen and fibronectin and quantification presented as area fraction of whole images (triplicates per experiment; 5 independent experiments). ( C ) Wound scratch cell migration assay of siRNA-transfected HDLEC immediately after injury, at 24 hr and 48 hrs afterward. ( D ) 3D tube formation networks in response to growth factors imaged 24 hrs post plating. ( E ) Annexin V apoptosis assay of siRNA-transfected HDLEC either untreated or following staurosporine treatment, quantification of total AnnexinV-positive cell populations and mean intensity of AnnexinV and propidium iodide of 3 independent experiments. Data presented as means±SEM. * p

    Article Snippet: Cell Adhesion Assay 96-wells plates were coated with 1% BSA, 10 µg/cm2 collagen or 10 µg/ml bovine plasma fibronectin (Sigma) diluted in calcium/magnesium-free PBS overnight.

    Techniques: In Vitro, Proliferation Assay, Cell Adhesion Assay, Transfection, Cell Migration Assay, Apoptosis Assay

    In vitro experiment demonstrating that AtCHIP is an E3 ligase. Ub, Ubiquitin; E1, ubiquitin-activating enzyme; E2, ubiquitinconjugating enzyme.

    Journal: Plant Physiology

    Article Title: AtCHIP, a U-Box-Containing E3 Ubiquitin Ligase, Plays a Critical Role in Temperature Stress Tolerance in Arabidopsis 1

    doi: 10.1104/pp.103.020800

    Figure Lengend Snippet: In vitro experiment demonstrating that AtCHIP is an E3 ligase. Ub, Ubiquitin; E1, ubiquitin-activating enzyme; E2, ubiquitinconjugating enzyme.

    Article Snippet: The reaction mixture included rabbit E1 (Calbiochem-Novabiochem, San Diego), human ( Homo sapiens ) E2 (UbcH5a), and bovine ubiquitin (Sigma, St. Louis).

    Techniques: In Vitro

    Colony-forming assay to assess cytotoxicity of 6-subsituted pyrrolo[2,3-d]pyrimidine analogues. IGROV1 EOC (~10 000) cells were plated in 100 mm dishes in complete folate-free RPMI 1640 (pH 7.2), 10% dialyzed FBS, and 25 nM LCV. After 24 h, cells were treated with PMX, 1 , or 11 at varying concentrations (0.1 to 20 μ M) in complete folate-free RPMI 1640 adjusted to pH 6.8 and supplemented with 25 nM LCV. After an additional 24 h, cells were rinsed with PBS, then incubated in complete folate-free RPMI 1640 supplemented with 25 nM LCV (pH 7.2–7.4) for 10 days. Colonies were stained with methylene blue; colony numbers were electronically counted, and results were normalized to the controls. Plots show mean ± SE values, representative of triplicate experiments. IC 50 values ( μ M) were as follows: PMX, > 20 μM; 11, 0.27 μ M, 1, 0.20 μ M.

    Journal: Journal of medicinal chemistry

    Article Title: Fluorine-Substituted Pyrrolo[2,3-d]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis

    doi: 10.1021/acs.jmedchem.8b00408

    Figure Lengend Snippet: Colony-forming assay to assess cytotoxicity of 6-subsituted pyrrolo[2,3-d]pyrimidine analogues. IGROV1 EOC (~10 000) cells were plated in 100 mm dishes in complete folate-free RPMI 1640 (pH 7.2), 10% dialyzed FBS, and 25 nM LCV. After 24 h, cells were treated with PMX, 1 , or 11 at varying concentrations (0.1 to 20 μ M) in complete folate-free RPMI 1640 adjusted to pH 6.8 and supplemented with 25 nM LCV. After an additional 24 h, cells were rinsed with PBS, then incubated in complete folate-free RPMI 1640 supplemented with 25 nM LCV (pH 7.2–7.4) for 10 days. Colonies were stained with methylene blue; colony numbers were electronically counted, and results were normalized to the controls. Plots show mean ± SE values, representative of triplicate experiments. IC 50 values ( μ M) were as follows: PMX, > 20 μM; 11, 0.27 μ M, 1, 0.20 μ M.

    Article Snippet: Prior to the cell proliferation assays (see below), RT16 and D4 cells were cultured in complete folate-free RPMI 1640 (without added folate), plus 10% dialyzed fetal bovine serum (FBS) (Sigma-Aldrich) and penicillin/streptomycin for 3 days.

    Techniques: Incubation, Staining

    TDA Compliance. Score distributions of the target and decoy N-glycopeptide matches from the mixture of bovine fetuin, lactoferrin, and kappa casein. Data includes both 20 and 40% ACN SPE fractions. a . Without self-consistency. b . With self-consistency.

    Journal: Analytical chemistry

    Article Title: Automated assignments of N- and O-site specific glycosylation with extensive glycan heterogeneity of glycoprotein mixtures

    doi: 10.1021/ac4006556

    Figure Lengend Snippet: TDA Compliance. Score distributions of the target and decoy N-glycopeptide matches from the mixture of bovine fetuin, lactoferrin, and kappa casein. Data includes both 20 and 40% ACN SPE fractions. a . Without self-consistency. b . With self-consistency.

    Article Snippet: Pronase E proteases, cyanogen bromide (CNBr) activated sepharose 4B (S4B) beads, bovine pancreatic ribonuclease, bovine lactoferrin, bovine kappa casein and bovine fetuin were obtained from Sigma Aldrich (St. Louis, MO).

    Techniques:

    Parachlamydiaceae -induced cell death in S2 cells is accompanied by apoptosis-like morphological and nuclear changes and DNA fragmentation. Panel (A) illustrates morphological and nuclear changes that were observed in S2 cells in response to infection with Parachlamydiaceae , but not Simkania . Insect cells were infected with S. negevensis , Pa. acanthamoebae , or P. amoebophila (MOI 2.5). At 7 h p.i. DNA was stained with DAPI (blue). Untreated cells and cells treated with the apoptosis inducer ActD (7 h) are shown for comparison. The bar corresponds to 10 µm. In (B) the detection of DNA fragmentation by TUNEL staining in S2 cells infected with Parachlamydiaceae is shown. S2 cells were either left untreated, incubated with ActD (10 h), or infected with Pa. acanthamoebae or P. amoebophila (MOI 2.5, 10 h). Bacteria were detected by immunostaining using antibodies raised against purified bacteria (red). TUNEL-positive nuclei are shown in green. Two additional controls were included, a negative control where TdT was omitted from the TUNEL reaction mixture and a positive control where cells were preincubated with DNase I to experimentally introduce DNA double strand breaks in all (also non-apoptotic) cells. Note that apart from this control, TUNEL-positive cells typically display other characteristic features of apoptotic cells, such as condensed and fragmented nuclei and formation of apoptotic bodies. After infection, TUNEL-positive cells were also frequently associated with bacteria. The bar indicates 10 µm.

    Journal: PLoS ONE

    Article Title: Lack of Effective Anti-Apoptotic Activities Restricts Growth of Parachlamydiaceae in Insect Cells

    doi: 10.1371/journal.pone.0029565

    Figure Lengend Snippet: Parachlamydiaceae -induced cell death in S2 cells is accompanied by apoptosis-like morphological and nuclear changes and DNA fragmentation. Panel (A) illustrates morphological and nuclear changes that were observed in S2 cells in response to infection with Parachlamydiaceae , but not Simkania . Insect cells were infected with S. negevensis , Pa. acanthamoebae , or P. amoebophila (MOI 2.5). At 7 h p.i. DNA was stained with DAPI (blue). Untreated cells and cells treated with the apoptosis inducer ActD (7 h) are shown for comparison. The bar corresponds to 10 µm. In (B) the detection of DNA fragmentation by TUNEL staining in S2 cells infected with Parachlamydiaceae is shown. S2 cells were either left untreated, incubated with ActD (10 h), or infected with Pa. acanthamoebae or P. amoebophila (MOI 2.5, 10 h). Bacteria were detected by immunostaining using antibodies raised against purified bacteria (red). TUNEL-positive nuclei are shown in green. Two additional controls were included, a negative control where TdT was omitted from the TUNEL reaction mixture and a positive control where cells were preincubated with DNase I to experimentally introduce DNA double strand breaks in all (also non-apoptotic) cells. Note that apart from this control, TUNEL-positive cells typically display other characteristic features of apoptotic cells, such as condensed and fragmented nuclei and formation of apoptotic bodies. After infection, TUNEL-positive cells were also frequently associated with bacteria. The bar indicates 10 µm.

    Article Snippet: In case of the TUNEL positive control, cells were pre-incubated with DNase I (Sigma-Aldrich; 100 U/ml in 50 mM Tris/HCl, 1 mg/ml BSA, pH 7.5) for 10 min. For the negative control, cells were incubated in labelling solution devoid of the enzyme terminal deoxynucleotidyl transferase (TdT).

    Techniques: Infection, Staining, TUNEL Assay, Incubation, Immunostaining, Purification, Negative Control, Positive Control, Introduce

    Effect of HPβCD treatment on Npc1 −/− microglia. ( A ) Representative FACS analysis of 7-week-old Npc1 +/+ and Npc1 −/− mice injected twice a week with either phosphate buffered saline (PBS) or HPβCD starting at day of life 7. Cell size (FSC-A) and granularity (SSC-A) returned toward normal in the HPβCD compared to PBS treated mutant mice. ( B ) IBA1 staining (green) in the cerebellum, thalamus and frontal cortex of 7-week-old Npc1 +/+ and Npc1 −/− mice treated with either PBS or HPβCD is consistent with a less activated morphology in the HPβCD treated mice. Scale bar: 20 µm. ( C ) IBA1 (green), CD68 (red) double staining in the thalamus tissue from 7-week-old Npc1 +/+ and Npc1 −/− mice treated with either PBS or HPβCD demonstrates decreased microglial activation in HPβCD treated Npc1 −/− mice. Scale bar: 20 µm. ( D ) FACS analysis of CD11b, CX 3 CR1, unesterified cholesterol and GM1 levels in microglia from Npc1 +/+ treated with PBS (red line) and from Npc1 −/− treated with either PBS (light blue shading) or HPβCD (dark blue line). ( E ) Total ROS production, ascertained by DCFDA staining of isolated microglia from 7-week old control and mutant mice, confirms decreased ROS in HPβCD treated mutant mice. Results are from three independent experiments with ≥3 samples per experiment. Each sample consisted of 10 000 microglial cells. ( F ) Microglial gene expression changes comparing Npc1 −/− mice treated with PBS (dark blue) or HPβCD (light blue) versus Npc1 +/+ injected with PBS and HPβCD. The 207 genes with padj

    Journal: Human Molecular Genetics

    Article Title: Microglia activation in Niemann–Pick disease, type C1 is amendable to therapeutic intervention

    doi: 10.1093/hmg/ddy112

    Figure Lengend Snippet: Effect of HPβCD treatment on Npc1 −/− microglia. ( A ) Representative FACS analysis of 7-week-old Npc1 +/+ and Npc1 −/− mice injected twice a week with either phosphate buffered saline (PBS) or HPβCD starting at day of life 7. Cell size (FSC-A) and granularity (SSC-A) returned toward normal in the HPβCD compared to PBS treated mutant mice. ( B ) IBA1 staining (green) in the cerebellum, thalamus and frontal cortex of 7-week-old Npc1 +/+ and Npc1 −/− mice treated with either PBS or HPβCD is consistent with a less activated morphology in the HPβCD treated mice. Scale bar: 20 µm. ( C ) IBA1 (green), CD68 (red) double staining in the thalamus tissue from 7-week-old Npc1 +/+ and Npc1 −/− mice treated with either PBS or HPβCD demonstrates decreased microglial activation in HPβCD treated Npc1 −/− mice. Scale bar: 20 µm. ( D ) FACS analysis of CD11b, CX 3 CR1, unesterified cholesterol and GM1 levels in microglia from Npc1 +/+ treated with PBS (red line) and from Npc1 −/− treated with either PBS (light blue shading) or HPβCD (dark blue line). ( E ) Total ROS production, ascertained by DCFDA staining of isolated microglia from 7-week old control and mutant mice, confirms decreased ROS in HPβCD treated mutant mice. Results are from three independent experiments with ≥3 samples per experiment. Each sample consisted of 10 000 microglial cells. ( F ) Microglial gene expression changes comparing Npc1 −/− mice treated with PBS (dark blue) or HPβCD (light blue) versus Npc1 +/+ injected with PBS and HPβCD. The 207 genes with padj

    Article Snippet: Brain were removed without the spinal cord and suspended and triturated in ice-cold FACS buffer [PBS containing 0.5% BSA (Sigma)] using syringe plunger.

    Techniques: FACS, Mouse Assay, Injection, Mutagenesis, Staining, Double Staining, Activation Assay, Isolation, Expressing