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  • 94
    Thermo Fisher confluency
    Elevated lysosomal activity results in enhanced fast protein degradation in Grn −/− MEF. a Turnover of 35 S-methionine radiolabeled proteins. MEF at 70–80% <t>confluency</t> were metabolically pulse-labeled with 35 S-methionine/cysteine for 1 h, followed by indicated chase periods. Radioactivity of 35 S- labeled proteins at chase time point 0 h was set to 100% and remaining radioactive-labeled proteins at later chase points were normalized to the initial radioactivity at time point 0 h. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare Grn −/− to Grn +/+ MEF ( n = 5), (*, p
    Confluency, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 25572 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    MatTek confluency
    Elevated lysosomal activity results in enhanced fast protein degradation in Grn −/− MEF. a Turnover of 35 S-methionine radiolabeled proteins. MEF at 70–80% <t>confluency</t> were metabolically pulse-labeled with 35 S-methionine/cysteine for 1 h, followed by indicated chase periods. Radioactivity of 35 S- labeled proteins at chase time point 0 h was set to 100% and remaining radioactive-labeled proteins at later chase points were normalized to the initial radioactivity at time point 0 h. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare Grn −/− to Grn +/+ MEF ( n = 5), (*, p
    Confluency, supplied by MatTek, used in various techniques. Bioz Stars score: 92/100, based on 397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    SPL Life Sciences confluency
    Elevated lysosomal activity results in enhanced fast protein degradation in Grn −/− MEF. a Turnover of 35 S-methionine radiolabeled proteins. MEF at 70–80% <t>confluency</t> were metabolically pulse-labeled with 35 S-methionine/cysteine for 1 h, followed by indicated chase periods. Radioactivity of 35 S- labeled proteins at chase time point 0 h was set to 100% and remaining radioactive-labeled proteins at later chase points were normalized to the initial radioactivity at time point 0 h. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare Grn −/− to Grn +/+ MEF ( n = 5), (*, p
    Confluency, supplied by SPL Life Sciences, used in various techniques. Bioz Stars score: 90/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Starlab confluency
    Elevated lysosomal activity results in enhanced fast protein degradation in Grn −/− MEF. a Turnover of 35 S-methionine radiolabeled proteins. MEF at 70–80% <t>confluency</t> were metabolically pulse-labeled with 35 S-methionine/cysteine for 1 h, followed by indicated chase periods. Radioactivity of 35 S- labeled proteins at chase time point 0 h was set to 100% and remaining radioactive-labeled proteins at later chase points were normalized to the initial radioactivity at time point 0 h. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare Grn −/− to Grn +/+ MEF ( n = 5), (*, p
    Confluency, supplied by Starlab, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore confluency
    Adhesion of melanoma cells to interendothelial junctions. RBECs were grown on 8 µm pore size filter inserts. After reaching <t>confluency</t> A2058 cells were plated into the upper chamber and left for 5 h. Samples were fixed and 60–70 nm sections were prepared for electron microscopy. Arrow indicates interendothelial cell contact site. M = melanoma cell, E = endothelial cell. Scale bar = 10 µm.
    Confluency, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 7467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Avantor confluency
    MAbs KU42.33C and KU43.13A are directed against distinct epitopes on BxPC-3 human pancreatic cancer cells BxPC-3 cells were grown in 96-well plates to near <t>confluency.</t> MAb KU42.33C was added in doubling dilutions at highest concentration of 25 μg/ml for 1 hour on ice. After washing unbound antibodies, increasing concentrations of mAb KU42.33C or mAb KU43.13A were added and the total level of bound antibodies were determined as described in Materials and Method section. Data is presented as mean absorbance at 450 nm ± SD.
    Confluency, supplied by Avantor, used in various techniques. Bioz Stars score: 93/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson confluency
    Knockdown of GNB4 inhibits proliferation and induces cell cycle arrest and apoptosis. a and b , 182 R -6 and TAM R -1 cells grown to 80% <t>confluency</t> were transiently transfected with either 30 nM GNB4 siRNA or 30 nM negative control siRNA; 24 h after transfection, the cells were replated in 96-well plate; the MTT assay was performed as described in “Methods”; 72 h (TAM R -1) or 96 h (182 R -6) after transfection, the cells were harvested for cell cycle and apoptosis analyses. Asterisk indicates p
    Confluency, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 987 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Biocoat confluency
    Knockdown of GNB4 inhibits proliferation and induces cell cycle arrest and apoptosis. a and b , 182 R -6 and TAM R -1 cells grown to 80% <t>confluency</t> were transiently transfected with either 30 nM GNB4 siRNA or 30 nM negative control siRNA; 24 h after transfection, the cells were replated in 96-well plate; the MTT assay was performed as described in “Methods”; 72 h (TAM R -1) or 96 h (182 R -6) after transfection, the cells were harvested for cell cycle and apoptosis analyses. Asterisk indicates p
    Confluency, supplied by Biocoat, used in various techniques. Bioz Stars score: 93/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical confluency
    Knockdown of GNB4 inhibits proliferation and induces cell cycle arrest and apoptosis. a and b , 182 R -6 and TAM R -1 cells grown to 80% <t>confluency</t> were transiently transfected with either 30 nM GNB4 siRNA or 30 nM negative control siRNA; 24 h after transfection, the cells were replated in 96-well plate; the MTT assay was performed as described in “Methods”; 72 h (TAM R -1) or 96 h (182 R -6) after transfection, the cells were harvested for cell cycle and apoptosis analyses. Asterisk indicates p
    Confluency, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc confluency
    Knockdown of GNB4 inhibits proliferation and induces cell cycle arrest and apoptosis. a and b , 182 R -6 and TAM R -1 cells grown to 80% <t>confluency</t> were transiently transfected with either 30 nM GNB4 siRNA or 30 nM negative control siRNA; 24 h after transfection, the cells were replated in 96-well plate; the MTT assay was performed as described in “Methods”; 72 h (TAM R -1) or 96 h (182 R -6) after transfection, the cells were harvested for cell cycle and apoptosis analyses. Asterisk indicates p
    Confluency, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cellgro confluency
    Knockdown of GNB4 inhibits proliferation and induces cell cycle arrest and apoptosis. a and b , 182 R -6 and TAM R -1 cells grown to 80% <t>confluency</t> were transiently transfected with either 30 nM GNB4 siRNA or 30 nM negative control siRNA; 24 h after transfection, the cells were replated in 96-well plate; the MTT assay was performed as described in “Methods”; 72 h (TAM R -1) or 96 h (182 R -6) after transfection, the cells were harvested for cell cycle and apoptosis analyses. Asterisk indicates p
    Confluency, supplied by Cellgro, used in various techniques. Bioz Stars score: 92/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Corning Life Sciences confluency
    GSK3ß −/− mouse embryonic fibroblasts display reduced restitution in response to scrape-wounding as compared to GSK3ß wt/wt cells. Mouse embryonic fibroblasts (MEF) were prepared from GSK3ß −/− and GSK3ß wt/wt mice and grown to <t>confluency.</t> Cells were serum-starved overnight, standardized wounds were created using a razor blade and restitution was monitored over 24 h using serial microphotography. Data are expressed as median and interquartile range (IQR) of n = 9 independent wounds per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p
    Confluency, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 1197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Eppendorf AG confluency
    Pro-cathepsin D is secreted from COG subunit KO cells but not WT cells . Cells at 80–100% <t>confluency</t> were placed in chemically defined serum free media. Media was collected at 24 h and then analyzed by western blot for Cathepsin D.
    Confluency, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 92/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Essen Bioscience confluency
    CFI-400945 affected tumor cell ability to reach <t>confluency</t> and had an impact on cell viability Significant delay in reaching confluency was observed when MON, G401, BT-12 and DAOY cells were treated with increasing concentrations of CFI-400945 starting at 31.25nM. Cells were plated at a density of 6,000 cells/well. (A) MON MRT cells (FC=1.56, p-value=0.004); (B) G401 RTK cells (FC=1.78, p-value=0.004); (C) BT-12 AT/RT cells (FC=1.89, p-value=0.001) and (D) DAOY MB cells (FC=1.70, p-value=0.029); (E) Dose-response curves created from viability assays for three rhabdoid cell lines and one MB cell line treated with concentrations of CFI-400945 ranging from 1nM to 10μM. IC50 values were calculated: MON (5.13μM), BT-12 (3.73μM), G401 (3.79μM), DAOY (0.094μM).
    Confluency, supplied by Essen Bioscience, used in various techniques. Bioz Stars score: 92/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Euromedex confluency
    CFI-400945 affected tumor cell ability to reach <t>confluency</t> and had an impact on cell viability Significant delay in reaching confluency was observed when MON, G401, BT-12 and DAOY cells were treated with increasing concentrations of CFI-400945 starting at 31.25nM. Cells were plated at a density of 6,000 cells/well. (A) MON MRT cells (FC=1.56, p-value=0.004); (B) G401 RTK cells (FC=1.78, p-value=0.004); (C) BT-12 AT/RT cells (FC=1.89, p-value=0.001) and (D) DAOY MB cells (FC=1.70, p-value=0.029); (E) Dose-response curves created from viability assays for three rhabdoid cell lines and one MB cell line treated with concentrations of CFI-400945 ranging from 1nM to 10μM. IC50 values were calculated: MON (5.13μM), BT-12 (3.73μM), G401 (3.79μM), DAOY (0.094μM).
    Confluency, supplied by Euromedex, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific confluency
    Long-term non-invasive monitoring of GFP expression patterns. (A) Time course measurements of the fraction of fluorescent cell pixels (i), mean fluorescence intensity of cell pixels (ii) and mean image <t>confluency</t> (iii). Oct4-GiP mES cells were cultured in 6-well plates in three different media formulation (expansion in blue, spontaneous differentiation in black and directed differentiation in red). Each data point is the mean of three field of views per well, across three wells. Error bars are the standard deviation. (B) Augmented Fluorescence Images (AFI) of Oct4-GiP mES cells in 6-well plates cultured in different media formulations: (i) expansion, (ii) spontaneous differentiation and (iii) directed differentiation. Green represents fluorescent cell pixels (dark green is high expression, light green is low expression), red indicates non-expressing cell pixels and black is background (non-cell) pixels. The time corresponding to each image is shown in the insert at the top.
    Confluency, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare confluency
    Dcp1a is phosphorylated during early differentiation of 3T3-L1 preadipocytes. (A) The expression profile of endogenous Dcp1a during the early differentiation of 3T3-L1 preadipocytes. Two days after reaching <t>confluency,</t> cultures of 3T3-L1 preadipocytes were induced to differentiate with an induction cocktail for 0, 1, 2, 4, 8, and 16 h. Cell extracts were isolated, and western blot analysis for detection of Dcp1a protein was conducted using 30 µg of each sample. Tubulin served as the protein input control. (B) CIP treatment. Confluent cultures of 3T3-L1 preadipocytes were induced to differentiate for 0–16 h, and cell extracts were isolated and treated with CIP. Western blot analysis was performed as in panel A. (C) Two days after reaching confluency, 3T3-L1 preadipocytes were induced to differentiate with individual components of the induction cocktail (FBS, MIX, DEX, insulin) or with the induction cocktail (FMDI) or non-induced control (NI). After incubating for 2 h, cell extracts were analyzed as in panel A. (D) Two days after reaching confluency, 3T3-L1 preadipocytes were pretreated with either DMSO (vehicle control) or 20 µM U0126 for 30 min before induction and then were induced with FMDI for 0, 1, 4, and 8 h in DMSO or U0126. Cell extracts were isolated for western blot analysis using anti-Dcp1a, anti-phospho-ERK (p-ERK), and anti-total ERK (t-ERK). All experiments were independently repeated three to five times with similar results, one of which is shown here.
    Confluency, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GraphPad Prism Inc confluency
    Dcp1a is phosphorylated during early differentiation of 3T3-L1 preadipocytes. (A) The expression profile of endogenous Dcp1a during the early differentiation of 3T3-L1 preadipocytes. Two days after reaching <t>confluency,</t> cultures of 3T3-L1 preadipocytes were induced to differentiate with an induction cocktail for 0, 1, 2, 4, 8, and 16 h. Cell extracts were isolated, and western blot analysis for detection of Dcp1a protein was conducted using 30 µg of each sample. Tubulin served as the protein input control. (B) CIP treatment. Confluent cultures of 3T3-L1 preadipocytes were induced to differentiate for 0–16 h, and cell extracts were isolated and treated with CIP. Western blot analysis was performed as in panel A. (C) Two days after reaching confluency, 3T3-L1 preadipocytes were induced to differentiate with individual components of the induction cocktail (FBS, MIX, DEX, insulin) or with the induction cocktail (FMDI) or non-induced control (NI). After incubating for 2 h, cell extracts were analyzed as in panel A. (D) Two days after reaching confluency, 3T3-L1 preadipocytes were pretreated with either DMSO (vehicle control) or 20 µM U0126 for 30 min before induction and then were induced with FMDI for 0, 1, 4, and 8 h in DMSO or U0126. Cell extracts were isolated for western blot analysis using anti-Dcp1a, anti-phospho-ERK (p-ERK), and anti-total ERK (t-ERK). All experiments were independently repeated three to five times with similar results, one of which is shown here.
    Confluency, supplied by GraphPad Prism Inc, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Greiner Bio confluency
    Bacterial invasion and survival in non-phagocytic lung and brain cells with and without alcohol treatment. (A) Lung epithelial and (B) brain endothelial cells were grown to <t>confluency</t> in F12 cell culture media and co-cultured with B . thailandensis or B . vietnamiensis (MOI 1:10) for 3 h in media supplemented with 0.0% or 0.2% v/v alcohol. Extracellular bacteria were removed by washes X4 and antibiotic treatment for 2 h. Cells were lysed and viable bacteria recovered. Asterisks (*) represent statistical comparisons between alcohol treatment and (Non-Alcohol) control determined by one-way ANOVA. Bars represent average CFU with SEM. **, p ≤ .01; ***, p ≤ .001; ****, p ≤ 0.0001.
    Confluency, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 92/100, based on 502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Horizon Discovery confluency
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% <t>confluency.</t> Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Confluency, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 93/100, based on 676 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ibidi confluency
    Ift88 expedites cell migration in previously unciliated MDCK cells. (A) Ift88-i1 cells were grown to <t>confluency</t> for 2 days (unciliated stage, compare Fig 3 ) and subjected to wounding. Cells depleted of Ift88 by inducible shRNA (+Tet) migrate more slowly compared with non-induced control cells (-Tet). The leading edge is shown after 2h and 6h. Scale bars: 100μm. (B) Quantification reveals no significant reduction of migration speed for a control cell line: -Tet: 157.1 ±12.8 μm 2 /minute vs. +Tet: 144.8 ±10.6 μm 2 /minute (n.s.; p = 0.5). Migration speed is reduced in two independent cell lines after induced depletion of Ift88. Ift88-i1: -Tet: 133.9 ±7.0 μm 2 /minute vs. +Tet: 50.4 ±2.5 μm 2 /minute, p
    Confluency, supplied by ibidi, used in various techniques. Bioz Stars score: 93/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Iwaki America confluency
    Ift88 expedites cell migration in previously unciliated MDCK cells. (A) Ift88-i1 cells were grown to <t>confluency</t> for 2 days (unciliated stage, compare Fig 3 ) and subjected to wounding. Cells depleted of Ift88 by inducible shRNA (+Tet) migrate more slowly compared with non-induced control cells (-Tet). The leading edge is shown after 2h and 6h. Scale bars: 100μm. (B) Quantification reveals no significant reduction of migration speed for a control cell line: -Tet: 157.1 ±12.8 μm 2 /minute vs. +Tet: 144.8 ±10.6 μm 2 /minute (n.s.; p = 0.5). Migration speed is reduced in two independent cell lines after induced depletion of Ift88. Ift88-i1: -Tet: 133.9 ±7.0 μm 2 /minute vs. +Tet: 50.4 ±2.5 μm 2 /minute, p
    Confluency, supplied by Iwaki America, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Lonza confluency
    Lipid vacuole formation and gene expression for adipogenic differentiation. AdMSCs were allowed to reach <t>confluency</t> and were then exposed to 0.5% CSE at the moment of induction of adipogenic differentiation with supplemented medium and for 24 h after induction. ( A ) After 21 d in culture, the cells were stained with Oil Red O to detect lipid vacuoles (10× magnification). ( B ) The Oil Red O solution was eluted and the optical density was measured, indicating there were no significant differences in lipid vacuole formation after acute CSE exposure. ( C ) RNA was extracted from the cells and the gene expression of early ( PPARγ ), late ( LEP ), and abundant ( ADIPOQ ) markers was analysed with qPCR, revealing no significant differences after 3 d.
    Confluency, supplied by Lonza, used in various techniques. Bioz Stars score: 92/100, based on 836 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co confluency
    Lipid vacuole formation and gene expression for adipogenic differentiation. AdMSCs were allowed to reach <t>confluency</t> and were then exposed to 0.5% CSE at the moment of induction of adipogenic differentiation with supplemented medium and for 24 h after induction. ( A ) After 21 d in culture, the cells were stained with Oil Red O to detect lipid vacuoles (10× magnification). ( B ) The Oil Red O solution was eluted and the optical density was measured, indicating there were no significant differences in lipid vacuole formation after acute CSE exposure. ( C ) RNA was extracted from the cells and the gene expression of early ( PPARγ ), late ( LEP ), and abundant ( ADIPOQ ) markers was analysed with qPCR, revealing no significant differences after 3 d.
    Confluency, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Mirus Bio confluency
    Aβ peptides inhibit migration of hP1-CL cells. Cell migration was measured as cell movement on glass cover slips in polystyrene dishes at 37 °C and 5% CO 2 . A PDMS barrier to cell growth was created on the cover slip. When cells reached <t>confluency,</t> the barrier was peeled off. Images of the resulting, initially sharp confluent cell boundary were captured at 0.003 Hz over 5 hours. Top panel shows initial (t = 0 min, black dotted lines) and final time points (t = 240 min, red dotted lines) of cell migration. hP1-CL cell migration was blocked by 5 µM D-GsMTx4, as well as 10 pM concentrations of L and D Aβ(1-40). Bottom left panel summarizes the mean cell migration distances as a function of time. Bottom right panel is the mean migration rate of hP1-CL cells in presence and absence of 5 µM D-GsMTx4, and of 10 pM of the L or D forms of Aβ(1-40). Three independent experiments were averaged (SEM).
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    Image Search Results


    Elevated lysosomal activity results in enhanced fast protein degradation in Grn −/− MEF. a Turnover of 35 S-methionine radiolabeled proteins. MEF at 70–80% confluency were metabolically pulse-labeled with 35 S-methionine/cysteine for 1 h, followed by indicated chase periods. Radioactivity of 35 S- labeled proteins at chase time point 0 h was set to 100% and remaining radioactive-labeled proteins at later chase points were normalized to the initial radioactivity at time point 0 h. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare Grn −/− to Grn +/+ MEF ( n = 5), (*, p

    Journal: Molecular Neurodegeneration

    Article Title: Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice

    doi: 10.1186/s13024-018-0281-5

    Figure Lengend Snippet: Elevated lysosomal activity results in enhanced fast protein degradation in Grn −/− MEF. a Turnover of 35 S-methionine radiolabeled proteins. MEF at 70–80% confluency were metabolically pulse-labeled with 35 S-methionine/cysteine for 1 h, followed by indicated chase periods. Radioactivity of 35 S- labeled proteins at chase time point 0 h was set to 100% and remaining radioactive-labeled proteins at later chase points were normalized to the initial radioactivity at time point 0 h. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare Grn −/− to Grn +/+ MEF ( n = 5), (*, p

    Article Snippet: Metabolic labeling and protein turn over To analyze protein turnover, MEF at 70–80% of confluency were starved for 1 h in methionine-, cysteine- and serum-free minimal essential medium (Invitrogen) and subsequently metabolically pulse-labeled with 18.5 MBq 35 S-methionine/cysteine (Met-S35-label, Hartmann Analytic) in methionine-, cysteine- and serum free medium for 1 h, followed by indicated chase periods in the presence of a 5-fold excess of unlabeled methionine.

    Techniques: Activity Assay, Metabolic Labelling, Labeling, Radioactivity, Two Tailed Test

    Adhesion of melanoma cells to interendothelial junctions. RBECs were grown on 8 µm pore size filter inserts. After reaching confluency A2058 cells were plated into the upper chamber and left for 5 h. Samples were fixed and 60–70 nm sections were prepared for electron microscopy. Arrow indicates interendothelial cell contact site. M = melanoma cell, E = endothelial cell. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: Transmigration of Melanoma Cells through the Blood-Brain Barrier: Role of Endothelial Tight Junctions and Melanoma-Released Serine Proteases

    doi: 10.1371/journal.pone.0020758

    Figure Lengend Snippet: Adhesion of melanoma cells to interendothelial junctions. RBECs were grown on 8 µm pore size filter inserts. After reaching confluency A2058 cells were plated into the upper chamber and left for 5 h. Samples were fixed and 60–70 nm sections were prepared for electron microscopy. Arrow indicates interendothelial cell contact site. M = melanoma cell, E = endothelial cell. Scale bar = 10 µm.

    Article Snippet: After reaching confluency, the endothelial monolayer was supplied with 550 nM hydrocortisone, 250 µM CPT-cAMP (Sigma) and 17.5 µM RO-201724 (Roche) and placed into the wells of the CellZscope® instrument (nanoAnalytics) containing astrocyte conditioned medium.

    Techniques: Electron Microscopy

    Role of gelatinolytic serine proteases produced by melanoma cells. A, B: Melanoma cells were plated onto confluent monolayers of cerebral endothelial cells or into empty culture dishes in serum-free medium in the presence or absence of E64 or Pefabloc® and left for 5 h. Culture media were collected and cells were lysed in Triton X-114 containing buffer. Samples were electrophoresed in non-denaturing conditions and the gels were incubated in EDTA-containing buffer for 2 days. Proteolytic bands of culture media (A) or cell lysates (B) were visualized by Coomassie blue staining. C, D: RBEC were grown until confluency on 8 µm pore size filter inserts. Fluorescently labeled melanoma cells (C: A2058, D: B16/F10) were plated into the upper chamber in the presence or absence of Pefabloc® and left for 5 h. Cells from the upper chamber were removed using a cotton swab, and melanoma cells migrated through the endothelial cell layer and the pores of the filter were counted. N = 3, * = P

    Journal: PLoS ONE

    Article Title: Transmigration of Melanoma Cells through the Blood-Brain Barrier: Role of Endothelial Tight Junctions and Melanoma-Released Serine Proteases

    doi: 10.1371/journal.pone.0020758

    Figure Lengend Snippet: Role of gelatinolytic serine proteases produced by melanoma cells. A, B: Melanoma cells were plated onto confluent monolayers of cerebral endothelial cells or into empty culture dishes in serum-free medium in the presence or absence of E64 or Pefabloc® and left for 5 h. Culture media were collected and cells were lysed in Triton X-114 containing buffer. Samples were electrophoresed in non-denaturing conditions and the gels were incubated in EDTA-containing buffer for 2 days. Proteolytic bands of culture media (A) or cell lysates (B) were visualized by Coomassie blue staining. C, D: RBEC were grown until confluency on 8 µm pore size filter inserts. Fluorescently labeled melanoma cells (C: A2058, D: B16/F10) were plated into the upper chamber in the presence or absence of Pefabloc® and left for 5 h. Cells from the upper chamber were removed using a cotton swab, and melanoma cells migrated through the endothelial cell layer and the pores of the filter were counted. N = 3, * = P

    Article Snippet: After reaching confluency, the endothelial monolayer was supplied with 550 nM hydrocortisone, 250 µM CPT-cAMP (Sigma) and 17.5 µM RO-201724 (Roche) and placed into the wells of the CellZscope® instrument (nanoAnalytics) containing astrocyte conditioned medium.

    Techniques: Produced, Incubation, Staining, Labeling

    MAbs KU42.33C and KU43.13A are directed against distinct epitopes on BxPC-3 human pancreatic cancer cells BxPC-3 cells were grown in 96-well plates to near confluency. MAb KU42.33C was added in doubling dilutions at highest concentration of 25 μg/ml for 1 hour on ice. After washing unbound antibodies, increasing concentrations of mAb KU42.33C or mAb KU43.13A were added and the total level of bound antibodies were determined as described in Materials and Method section. Data is presented as mean absorbance at 450 nm ± SD.

    Journal: Oncotarget

    Article Title: Development of novel monoclonal antibodies against CD109 overexpressed in human pancreatic cancer

    doi: 10.18632/oncotarget.25017

    Figure Lengend Snippet: MAbs KU42.33C and KU43.13A are directed against distinct epitopes on BxPC-3 human pancreatic cancer cells BxPC-3 cells were grown in 96-well plates to near confluency. MAb KU42.33C was added in doubling dilutions at highest concentration of 25 μg/ml for 1 hour on ice. After washing unbound antibodies, increasing concentrations of mAb KU42.33C or mAb KU43.13A were added and the total level of bound antibodies were determined as described in Materials and Method section. Data is presented as mean absorbance at 450 nm ± SD.

    Article Snippet: Briefly, BxPC-3 cancer cells were grown to near confluency in RPMI/10% FBS in Lab-Tek 8-well chamber slides (VWR, UK) or 96-well plates, respectively.

    Techniques: Concentration Assay

    Internalisation studies of novel mAbs KU42.33C and KU43.13A in BxPC-3 human pancreatic cancer cells determined by (A) immunofluorescence, and (B) ELISA. BxPC-3 cancer cells were grown to near confluency and incubated with purified antibodies (50 μg/ml) or control (PBS/1% BSA) at 4ºC for 1 h and subsequently at 37ºC for extra 30 min to allow internalisation. Cells were then fixed, permeabilised and incubated with FITC-conjugated or HRP-linked anti-mouse secondary antibodies for immunofluorescence staining (200x magnifications) and ELISA respectively. The anti-EGFR mAb HM43.16B. was used as a control (arrows: internalised antibody). ELISA results (B) are presented as mean absorbance ± SD.

    Journal: Oncotarget

    Article Title: Development of novel monoclonal antibodies against CD109 overexpressed in human pancreatic cancer

    doi: 10.18632/oncotarget.25017

    Figure Lengend Snippet: Internalisation studies of novel mAbs KU42.33C and KU43.13A in BxPC-3 human pancreatic cancer cells determined by (A) immunofluorescence, and (B) ELISA. BxPC-3 cancer cells were grown to near confluency and incubated with purified antibodies (50 μg/ml) or control (PBS/1% BSA) at 4ºC for 1 h and subsequently at 37ºC for extra 30 min to allow internalisation. Cells were then fixed, permeabilised and incubated with FITC-conjugated or HRP-linked anti-mouse secondary antibodies for immunofluorescence staining (200x magnifications) and ELISA respectively. The anti-EGFR mAb HM43.16B. was used as a control (arrows: internalised antibody). ELISA results (B) are presented as mean absorbance ± SD.

    Article Snippet: Briefly, BxPC-3 cancer cells were grown to near confluency in RPMI/10% FBS in Lab-Tek 8-well chamber slides (VWR, UK) or 96-well plates, respectively.

    Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Incubation, Purification, Staining

    Knockdown of GNB4 inhibits proliferation and induces cell cycle arrest and apoptosis. a and b , 182 R -6 and TAM R -1 cells grown to 80% confluency were transiently transfected with either 30 nM GNB4 siRNA or 30 nM negative control siRNA; 24 h after transfection, the cells were replated in 96-well plate; the MTT assay was performed as described in “Methods”; 72 h (TAM R -1) or 96 h (182 R -6) after transfection, the cells were harvested for cell cycle and apoptosis analyses. Asterisk indicates p

    Journal: BMC Cancer

    Article Title: A suppressive role of guanine nucleotide-binding protein subunit beta-4 inhibited by DNA methylation in the growth of anti-estrogen resistant breast cancer cells

    doi: 10.1186/s12885-018-4711-0

    Figure Lengend Snippet: Knockdown of GNB4 inhibits proliferation and induces cell cycle arrest and apoptosis. a and b , 182 R -6 and TAM R -1 cells grown to 80% confluency were transiently transfected with either 30 nM GNB4 siRNA or 30 nM negative control siRNA; 24 h after transfection, the cells were replated in 96-well plate; the MTT assay was performed as described in “Methods”; 72 h (TAM R -1) or 96 h (182 R -6) after transfection, the cells were harvested for cell cycle and apoptosis analyses. Asterisk indicates p

    Article Snippet: Cell cycle and apoptosis analyses 182R -6 or TAMR -1 cells stably expressing either GFP or GFP-GNB4 grown to 90% confluency were harvested for cell-cycle and apoptosis analyses that were performed with a BD FACSCanto™ II Flow Cytometer (BD Biosciences) using a GFP-Certified Nuclear-ID Red Cell Cycle Analysis Kit (Enzo) and an Annexin V-Cy3 Apoptosis Kit Plus (BioVision) according to the manufacturer’s instructions.

    Techniques: Transfection, Negative Control, MTT Assay

    The ectopic expression of GNB4 causes alterations in cell cycle and apoptosis. a and b , 182 R -6 and TAM R -1 cells stably expressing GNB4 or GFP grown to 90% confluency were subjected to cell cycle analysis using a GFP-Certified Nuclear-ID Red Cell Cycle Analysis Kit according to the manufacturer’s instructions. c and d , 182 R -6 and TAM R -1 cells stably expressing GNB4 or GFP grown to 90% confluency were subjected to apoptosis analysis using an Annexin V-Cy3 Apoptosis Kit Plus according to the manufacturer’s instructions. Asterisk indicates p

    Journal: BMC Cancer

    Article Title: A suppressive role of guanine nucleotide-binding protein subunit beta-4 inhibited by DNA methylation in the growth of anti-estrogen resistant breast cancer cells

    doi: 10.1186/s12885-018-4711-0

    Figure Lengend Snippet: The ectopic expression of GNB4 causes alterations in cell cycle and apoptosis. a and b , 182 R -6 and TAM R -1 cells stably expressing GNB4 or GFP grown to 90% confluency were subjected to cell cycle analysis using a GFP-Certified Nuclear-ID Red Cell Cycle Analysis Kit according to the manufacturer’s instructions. c and d , 182 R -6 and TAM R -1 cells stably expressing GNB4 or GFP grown to 90% confluency were subjected to apoptosis analysis using an Annexin V-Cy3 Apoptosis Kit Plus according to the manufacturer’s instructions. Asterisk indicates p

    Article Snippet: Cell cycle and apoptosis analyses 182R -6 or TAMR -1 cells stably expressing either GFP or GFP-GNB4 grown to 90% confluency were harvested for cell-cycle and apoptosis analyses that were performed with a BD FACSCanto™ II Flow Cytometer (BD Biosciences) using a GFP-Certified Nuclear-ID Red Cell Cycle Analysis Kit (Enzo) and an Annexin V-Cy3 Apoptosis Kit Plus (BioVision) according to the manufacturer’s instructions.

    Techniques: Expressing, Stable Transfection, Cell Cycle Assay

    GSK3ß −/− mouse embryonic fibroblasts display reduced restitution in response to scrape-wounding as compared to GSK3ß wt/wt cells. Mouse embryonic fibroblasts (MEF) were prepared from GSK3ß −/− and GSK3ß wt/wt mice and grown to confluency. Cells were serum-starved overnight, standardized wounds were created using a razor blade and restitution was monitored over 24 h using serial microphotography. Data are expressed as median and interquartile range (IQR) of n = 9 independent wounds per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p

    Journal: PLoS ONE

    Article Title: PI3K-Dependent GSK3ss(Ser9)-Phosphorylation Is Implicated in the Intestinal Epithelial Cell Wound-Healing Response

    doi: 10.1371/journal.pone.0026340

    Figure Lengend Snippet: GSK3ß −/− mouse embryonic fibroblasts display reduced restitution in response to scrape-wounding as compared to GSK3ß wt/wt cells. Mouse embryonic fibroblasts (MEF) were prepared from GSK3ß −/− and GSK3ß wt/wt mice and grown to confluency. Cells were serum-starved overnight, standardized wounds were created using a razor blade and restitution was monitored over 24 h using serial microphotography. Data are expressed as median and interquartile range (IQR) of n = 9 independent wounds per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p

    Article Snippet: Cells were grown to confluency in 6-well plates (Costar, Corning Inc, Acton, MA), starved overnight in serum-reduced media (1% FCS), and then standardized wounding was performed by multiple linear scraping with a P1000 pipet tip as described previously , , , , , .

    Techniques: Mouse Assay

    Wounding induces c-myc mRNA accumulation in IEC18 cell monolayers. Rat intestinal epithelial IEC18 cells were grown to confluency in 6-well-plates, serum-starved overnight, and cell monolayers were then wounded by multiple scraping. At the indicated time points, total cell RNA extracts were prepared and analysed for c-myc mRNA accumulation normalized to GAPDH control via real-time RT-PCR using specific primers. mRNA accumulation relative to unwounded control is expressed as median and interquartile range (IQR) of n = 3 separate experiments, each with three separate wells per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p

    Journal: PLoS ONE

    Article Title: PI3K-Dependent GSK3ss(Ser9)-Phosphorylation Is Implicated in the Intestinal Epithelial Cell Wound-Healing Response

    doi: 10.1371/journal.pone.0026340

    Figure Lengend Snippet: Wounding induces c-myc mRNA accumulation in IEC18 cell monolayers. Rat intestinal epithelial IEC18 cells were grown to confluency in 6-well-plates, serum-starved overnight, and cell monolayers were then wounded by multiple scraping. At the indicated time points, total cell RNA extracts were prepared and analysed for c-myc mRNA accumulation normalized to GAPDH control via real-time RT-PCR using specific primers. mRNA accumulation relative to unwounded control is expressed as median and interquartile range (IQR) of n = 3 separate experiments, each with three separate wells per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p

    Article Snippet: Cells were grown to confluency in 6-well plates (Costar, Corning Inc, Acton, MA), starved overnight in serum-reduced media (1% FCS), and then standardized wounding was performed by multiple linear scraping with a P1000 pipet tip as described previously , , , , , .

    Techniques: Quantitative RT-PCR

    Wounding induces GSK3ß(Ser9) phosphorylation, ß-catenin accumulation and nuclear translocation in IEC18 cell monolayers. Rat intestinal epithelial IEC18 cells were grown to confluency in 6-well-plates, serum-starved overnight, and cell monolayers were then wounded by multiple scraping. (A) At the indicated time points, total cell protein extracts were prepared and analysed for GSK3ß signaling pathway activation via Western blot. (B) 30 minutes after wounding, cells were lysed, subcellular fractions of nuclear/cytoplasmic protein were prepared and analysed for phospho-GSK3ß(Ser9) and ß-catenin accumulation via Western blot. (A,B) Densitometry was performed on n = 3–4 different Western blots (each representative of an independent experiment) per condition and normalized to the respective loading controls (GAPDH, SOD, PARP). Protein expression is expressed as -fold protein induction relative to the respective unwounded control. Data are expressed as means ± S.D. of 3–4 different experiments per condition. Statistical analysis was performed by the two-tailed Student's t test (*p

    Journal: PLoS ONE

    Article Title: PI3K-Dependent GSK3ss(Ser9)-Phosphorylation Is Implicated in the Intestinal Epithelial Cell Wound-Healing Response

    doi: 10.1371/journal.pone.0026340

    Figure Lengend Snippet: Wounding induces GSK3ß(Ser9) phosphorylation, ß-catenin accumulation and nuclear translocation in IEC18 cell monolayers. Rat intestinal epithelial IEC18 cells were grown to confluency in 6-well-plates, serum-starved overnight, and cell monolayers were then wounded by multiple scraping. (A) At the indicated time points, total cell protein extracts were prepared and analysed for GSK3ß signaling pathway activation via Western blot. (B) 30 minutes after wounding, cells were lysed, subcellular fractions of nuclear/cytoplasmic protein were prepared and analysed for phospho-GSK3ß(Ser9) and ß-catenin accumulation via Western blot. (A,B) Densitometry was performed on n = 3–4 different Western blots (each representative of an independent experiment) per condition and normalized to the respective loading controls (GAPDH, SOD, PARP). Protein expression is expressed as -fold protein induction relative to the respective unwounded control. Data are expressed as means ± S.D. of 3–4 different experiments per condition. Statistical analysis was performed by the two-tailed Student's t test (*p

    Article Snippet: Cells were grown to confluency in 6-well plates (Costar, Corning Inc, Acton, MA), starved overnight in serum-reduced media (1% FCS), and then standardized wounding was performed by multiple linear scraping with a P1000 pipet tip as described previously , , , , , .

    Techniques: Translocation Assay, Activation Assay, Western Blot, Expressing, Two Tailed Test

    Wounding strongly induces TCF/LEF-dependent gene transcription in IEC18 cell monolayers as compared to control-treated cells. IEC18 cells were grown to confluency, transfected with a TOP/FOP-reporter plasmid (expressing the luciferase gene under the TCF/LEF promoter) and then wounded by multiple scraping. Cell extracts were prepared after 13 h and 24 h, and luciferase activity was determined in cell lysates and normalized for protein content in the respective wells. The GSK3ß inhibitor Lithium Chloride (LiCl, 20 mmol/l) was used as positive control. Data are expressed as median and interquartile range (IQR) of n = 3–4 independent experiments, each with three separate wounds/dishes per condition, and luciferase activity was adjusted for total protein content of the respective cell lysates. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p

    Journal: PLoS ONE

    Article Title: PI3K-Dependent GSK3ss(Ser9)-Phosphorylation Is Implicated in the Intestinal Epithelial Cell Wound-Healing Response

    doi: 10.1371/journal.pone.0026340

    Figure Lengend Snippet: Wounding strongly induces TCF/LEF-dependent gene transcription in IEC18 cell monolayers as compared to control-treated cells. IEC18 cells were grown to confluency, transfected with a TOP/FOP-reporter plasmid (expressing the luciferase gene under the TCF/LEF promoter) and then wounded by multiple scraping. Cell extracts were prepared after 13 h and 24 h, and luciferase activity was determined in cell lysates and normalized for protein content in the respective wells. The GSK3ß inhibitor Lithium Chloride (LiCl, 20 mmol/l) was used as positive control. Data are expressed as median and interquartile range (IQR) of n = 3–4 independent experiments, each with three separate wounds/dishes per condition, and luciferase activity was adjusted for total protein content of the respective cell lysates. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p

    Article Snippet: Cells were grown to confluency in 6-well plates (Costar, Corning Inc, Acton, MA), starved overnight in serum-reduced media (1% FCS), and then standardized wounding was performed by multiple linear scraping with a P1000 pipet tip as described previously , , , , , .

    Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Positive Control

    Pro-cathepsin D is secreted from COG subunit KO cells but not WT cells . Cells at 80–100% confluency were placed in chemically defined serum free media. Media was collected at 24 h and then analyzed by western blot for Cathepsin D.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: COG Complex Complexities: Detailed Characterization of a Complete Set of HEK293T Cells Lacking Individual COG Subunits

    doi: 10.3389/fcell.2016.00023

    Figure Lengend Snippet: Pro-cathepsin D is secreted from COG subunit KO cells but not WT cells . Cells at 80–100% confluency were placed in chemically defined serum free media. Media was collected at 24 h and then analyzed by western blot for Cathepsin D.

    Article Snippet: Flow cytometry Cells were grown to 80–100% confluency then resuspened in ice cold 0.1% BSA by gentle pipetting and placed in an Eppendorf tube.

    Techniques: Western Blot

    CFI-400945 affected tumor cell ability to reach confluency and had an impact on cell viability Significant delay in reaching confluency was observed when MON, G401, BT-12 and DAOY cells were treated with increasing concentrations of CFI-400945 starting at 31.25nM. Cells were plated at a density of 6,000 cells/well. (A) MON MRT cells (FC=1.56, p-value=0.004); (B) G401 RTK cells (FC=1.78, p-value=0.004); (C) BT-12 AT/RT cells (FC=1.89, p-value=0.001) and (D) DAOY MB cells (FC=1.70, p-value=0.029); (E) Dose-response curves created from viability assays for three rhabdoid cell lines and one MB cell line treated with concentrations of CFI-400945 ranging from 1nM to 10μM. IC50 values were calculated: MON (5.13μM), BT-12 (3.73μM), G401 (3.79μM), DAOY (0.094μM).

    Journal: Oncotarget

    Article Title: Inhibition of polo-like kinase 4 (PLK4): a new therapeutic option for rhabdoid tumors and pediatric medulloblastoma

    doi: 10.18632/oncotarget.22704

    Figure Lengend Snippet: CFI-400945 affected tumor cell ability to reach confluency and had an impact on cell viability Significant delay in reaching confluency was observed when MON, G401, BT-12 and DAOY cells were treated with increasing concentrations of CFI-400945 starting at 31.25nM. Cells were plated at a density of 6,000 cells/well. (A) MON MRT cells (FC=1.56, p-value=0.004); (B) G401 RTK cells (FC=1.78, p-value=0.004); (C) BT-12 AT/RT cells (FC=1.89, p-value=0.001) and (D) DAOY MB cells (FC=1.70, p-value=0.029); (E) Dose-response curves created from viability assays for three rhabdoid cell lines and one MB cell line treated with concentrations of CFI-400945 ranging from 1nM to 10μM. IC50 values were calculated: MON (5.13μM), BT-12 (3.73μM), G401 (3.79μM), DAOY (0.094μM).

    Article Snippet: Confluency was monitored for 48 hours by taking phase contrast images every 2 hours and measured using an IncuCyte ZOOM system (Essen BioScience, USA).

    Techniques:

    Long-term non-invasive monitoring of GFP expression patterns. (A) Time course measurements of the fraction of fluorescent cell pixels (i), mean fluorescence intensity of cell pixels (ii) and mean image confluency (iii). Oct4-GiP mES cells were cultured in 6-well plates in three different media formulation (expansion in blue, spontaneous differentiation in black and directed differentiation in red). Each data point is the mean of three field of views per well, across three wells. Error bars are the standard deviation. (B) Augmented Fluorescence Images (AFI) of Oct4-GiP mES cells in 6-well plates cultured in different media formulations: (i) expansion, (ii) spontaneous differentiation and (iii) directed differentiation. Green represents fluorescent cell pixels (dark green is high expression, light green is low expression), red indicates non-expressing cell pixels and black is background (non-cell) pixels. The time corresponding to each image is shown in the insert at the top.

    Journal: Biotechnology and Bioengineering

    Article Title: Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

    doi: 10.1002/bit.25115

    Figure Lengend Snippet: Long-term non-invasive monitoring of GFP expression patterns. (A) Time course measurements of the fraction of fluorescent cell pixels (i), mean fluorescence intensity of cell pixels (ii) and mean image confluency (iii). Oct4-GiP mES cells were cultured in 6-well plates in three different media formulation (expansion in blue, spontaneous differentiation in black and directed differentiation in red). Each data point is the mean of three field of views per well, across three wells. Error bars are the standard deviation. (B) Augmented Fluorescence Images (AFI) of Oct4-GiP mES cells in 6-well plates cultured in different media formulations: (i) expansion, (ii) spontaneous differentiation and (iii) directed differentiation. Green represents fluorescent cell pixels (dark green is high expression, light green is low expression), red indicates non-expressing cell pixels and black is background (non-cell) pixels. The time corresponding to each image is shown in the insert at the top.

    Article Snippet: Cell Culture Experiments for Confluency Monitoring Undifferentiated mESC were dissociated and inoculated onto 0.1% (w/v) gelatin-coated tissue culture 6-well plates (Fischer Scientific) at a density of 5 × 104 cells cm−2 in 2 mL of medium.

    Techniques: Expressing, Fluorescence, Cell Culture, Standard Deviation

    Summary of the proposed method PCM images were first segmented using local contrast thresholding and post hoc halo correction. Confluency could be determined directly from the outcome of the segmentation. Morphological analysis of cellular objects was carried out using convexity and shape factor as metrics. Cell density could be estimated using packing-corrected confluency, a metric based both on confluency and the mean distance between blob-like texture features. Finally, PCM segmentation was combined with fluorescence imaging data to enable the determination of temporal and spatial fluorescence patterns.

    Journal: Biotechnology and Bioengineering

    Article Title: Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

    doi: 10.1002/bit.25115

    Figure Lengend Snippet: Summary of the proposed method PCM images were first segmented using local contrast thresholding and post hoc halo correction. Confluency could be determined directly from the outcome of the segmentation. Morphological analysis of cellular objects was carried out using convexity and shape factor as metrics. Cell density could be estimated using packing-corrected confluency, a metric based both on confluency and the mean distance between blob-like texture features. Finally, PCM segmentation was combined with fluorescence imaging data to enable the determination of temporal and spatial fluorescence patterns.

    Article Snippet: Cell Culture Experiments for Confluency Monitoring Undifferentiated mESC were dissociated and inoculated onto 0.1% (w/v) gelatin-coated tissue culture 6-well plates (Fischer Scientific) at a density of 5 × 104 cells cm−2 in 2 mL of medium.

    Techniques: Fluorescence, Imaging

    Method for the segmentation of phase contrast microscopy (PCM) images. (A) (i) Cropped region of a mouse embryonic stem cell PCM image shortly after seeding. Insert shows a zoomed-in region with description of the key features of a typical PCM image including a halo artifact surrounding the cell and the lack of contrast between the background and the interior of the cell (ii) Cell contour detected (black line) after local contrast thresholding. A large portion of the pixels corresponding to the halo artifact are incorrectly classified as cell pixels. (iii) Cell contour detected (black line) after post-segmentation halo correction. It conforms to the actual contour of the cell. Scale bars are 10 µm. (B) F -score as a function of the ground truth image confluency. Closed symbols represent images with degraded quality due to condensation. (C) Examples of segmentation outcomes for mouse embryonic stem cells (mESC), Chinese hamster ovary cells (CHO) and neuroblastoma cells. For each example, the raw PCM image overlaid with the detected border is compared with the ground truth image (to the right of the raw image). True positives are yellow, false positives green, true negatives black, and false negatives red. Scale bars are 50 µm.

    Journal: Biotechnology and Bioengineering

    Article Title: Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

    doi: 10.1002/bit.25115

    Figure Lengend Snippet: Method for the segmentation of phase contrast microscopy (PCM) images. (A) (i) Cropped region of a mouse embryonic stem cell PCM image shortly after seeding. Insert shows a zoomed-in region with description of the key features of a typical PCM image including a halo artifact surrounding the cell and the lack of contrast between the background and the interior of the cell (ii) Cell contour detected (black line) after local contrast thresholding. A large portion of the pixels corresponding to the halo artifact are incorrectly classified as cell pixels. (iii) Cell contour detected (black line) after post-segmentation halo correction. It conforms to the actual contour of the cell. Scale bars are 10 µm. (B) F -score as a function of the ground truth image confluency. Closed symbols represent images with degraded quality due to condensation. (C) Examples of segmentation outcomes for mouse embryonic stem cells (mESC), Chinese hamster ovary cells (CHO) and neuroblastoma cells. For each example, the raw PCM image overlaid with the detected border is compared with the ground truth image (to the right of the raw image). True positives are yellow, false positives green, true negatives black, and false negatives red. Scale bars are 50 µm.

    Article Snippet: Cell Culture Experiments for Confluency Monitoring Undifferentiated mESC were dissociated and inoculated onto 0.1% (w/v) gelatin-coated tissue culture 6-well plates (Fischer Scientific) at a density of 5 × 104 cells cm−2 in 2 mL of medium.

    Techniques: Microscopy

    Cell density estimation of mESC cultures based on PCM images. (A) Relationship (adjusted r 2 = 0.89) between cell density (as measured after detachment) and culture confluency (determined using PHANTAST). (B) Changes in mean cell area during a typical mESC culture. Cells were counted using a live nuclear stain (Hoechst 33342). The insert shows the change in distance between nuclei during the same culture. (C) The relationship between cell density (as determined after detachment) and packing-corrected confluency (PCC), computed from the confluency and the distance to nearest nucleus-like feature using PHANTAST.

    Journal: Biotechnology and Bioengineering

    Article Title: Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

    doi: 10.1002/bit.25115

    Figure Lengend Snippet: Cell density estimation of mESC cultures based on PCM images. (A) Relationship (adjusted r 2 = 0.89) between cell density (as measured after detachment) and culture confluency (determined using PHANTAST). (B) Changes in mean cell area during a typical mESC culture. Cells were counted using a live nuclear stain (Hoechst 33342). The insert shows the change in distance between nuclei during the same culture. (C) The relationship between cell density (as determined after detachment) and packing-corrected confluency (PCC), computed from the confluency and the distance to nearest nucleus-like feature using PHANTAST.

    Article Snippet: Cell Culture Experiments for Confluency Monitoring Undifferentiated mESC were dissociated and inoculated onto 0.1% (w/v) gelatin-coated tissue culture 6-well plates (Fischer Scientific) at a density of 5 × 104 cells cm−2 in 2 mL of medium.

    Techniques: Staining, Periodic Counter-current Chromatography

    Monitoring of mESC cultures. (A) (i) Time course study of the effect of medium exchange on confluency. Twenty random PCM images per well (of a 6-well plate), at 10× magnification, were used for confluency determination using PHANTAST. Confluency is determined with high precision in

    Journal: Biotechnology and Bioengineering

    Article Title: Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

    doi: 10.1002/bit.25115

    Figure Lengend Snippet: Monitoring of mESC cultures. (A) (i) Time course study of the effect of medium exchange on confluency. Twenty random PCM images per well (of a 6-well plate), at 10× magnification, were used for confluency determination using PHANTAST. Confluency is determined with high precision in

    Article Snippet: Cell Culture Experiments for Confluency Monitoring Undifferentiated mESC were dissociated and inoculated onto 0.1% (w/v) gelatin-coated tissue culture 6-well plates (Fischer Scientific) at a density of 5 × 104 cells cm−2 in 2 mL of medium.

    Techniques:

    Dcp1a is phosphorylated during early differentiation of 3T3-L1 preadipocytes. (A) The expression profile of endogenous Dcp1a during the early differentiation of 3T3-L1 preadipocytes. Two days after reaching confluency, cultures of 3T3-L1 preadipocytes were induced to differentiate with an induction cocktail for 0, 1, 2, 4, 8, and 16 h. Cell extracts were isolated, and western blot analysis for detection of Dcp1a protein was conducted using 30 µg of each sample. Tubulin served as the protein input control. (B) CIP treatment. Confluent cultures of 3T3-L1 preadipocytes were induced to differentiate for 0–16 h, and cell extracts were isolated and treated with CIP. Western blot analysis was performed as in panel A. (C) Two days after reaching confluency, 3T3-L1 preadipocytes were induced to differentiate with individual components of the induction cocktail (FBS, MIX, DEX, insulin) or with the induction cocktail (FMDI) or non-induced control (NI). After incubating for 2 h, cell extracts were analyzed as in panel A. (D) Two days after reaching confluency, 3T3-L1 preadipocytes were pretreated with either DMSO (vehicle control) or 20 µM U0126 for 30 min before induction and then were induced with FMDI for 0, 1, 4, and 8 h in DMSO or U0126. Cell extracts were isolated for western blot analysis using anti-Dcp1a, anti-phospho-ERK (p-ERK), and anti-total ERK (t-ERK). All experiments were independently repeated three to five times with similar results, one of which is shown here.

    Journal: PLoS ONE

    Article Title: Phosphorylation of mRNA Decapping Protein Dcp1a by the ERK Signaling Pathway during Early Differentiation of 3T3-L1 Preadipocytes

    doi: 10.1371/journal.pone.0061697

    Figure Lengend Snippet: Dcp1a is phosphorylated during early differentiation of 3T3-L1 preadipocytes. (A) The expression profile of endogenous Dcp1a during the early differentiation of 3T3-L1 preadipocytes. Two days after reaching confluency, cultures of 3T3-L1 preadipocytes were induced to differentiate with an induction cocktail for 0, 1, 2, 4, 8, and 16 h. Cell extracts were isolated, and western blot analysis for detection of Dcp1a protein was conducted using 30 µg of each sample. Tubulin served as the protein input control. (B) CIP treatment. Confluent cultures of 3T3-L1 preadipocytes were induced to differentiate for 0–16 h, and cell extracts were isolated and treated with CIP. Western blot analysis was performed as in panel A. (C) Two days after reaching confluency, 3T3-L1 preadipocytes were induced to differentiate with individual components of the induction cocktail (FBS, MIX, DEX, insulin) or with the induction cocktail (FMDI) or non-induced control (NI). After incubating for 2 h, cell extracts were analyzed as in panel A. (D) Two days after reaching confluency, 3T3-L1 preadipocytes were pretreated with either DMSO (vehicle control) or 20 µM U0126 for 30 min before induction and then were induced with FMDI for 0, 1, 4, and 8 h in DMSO or U0126. Cell extracts were isolated for western blot analysis using anti-Dcp1a, anti-phospho-ERK (p-ERK), and anti-total ERK (t-ERK). All experiments were independently repeated three to five times with similar results, one of which is shown here.

    Article Snippet: Two days after reaching confluency, 3T3-L1 cells were stimulated to undergo differentiation into adipocytes by replacing fresh medium with an induction cocktail, FMDI, that was comprised of 10% FBS (HyClone-Characterized), 0.5 mM MIX (Sigma-Aldrich), 5 µM DEX (Sigma-Aldrich), and 1.7 µM bovine insulin.

    Techniques: Expressing, Isolation, Western Blot

    Bacterial invasion and survival in non-phagocytic lung and brain cells with and without alcohol treatment. (A) Lung epithelial and (B) brain endothelial cells were grown to confluency in F12 cell culture media and co-cultured with B . thailandensis or B . vietnamiensis (MOI 1:10) for 3 h in media supplemented with 0.0% or 0.2% v/v alcohol. Extracellular bacteria were removed by washes X4 and antibiotic treatment for 2 h. Cells were lysed and viable bacteria recovered. Asterisks (*) represent statistical comparisons between alcohol treatment and (Non-Alcohol) control determined by one-way ANOVA. Bars represent average CFU with SEM. **, p ≤ .01; ***, p ≤ .001; ****, p ≤ 0.0001.

    Journal: PLoS ONE

    Article Title: A mouse model of binge alcohol consumption and Burkholderia infection

    doi: 10.1371/journal.pone.0208061

    Figure Lengend Snippet: Bacterial invasion and survival in non-phagocytic lung and brain cells with and without alcohol treatment. (A) Lung epithelial and (B) brain endothelial cells were grown to confluency in F12 cell culture media and co-cultured with B . thailandensis or B . vietnamiensis (MOI 1:10) for 3 h in media supplemented with 0.0% or 0.2% v/v alcohol. Extracellular bacteria were removed by washes X4 and antibiotic treatment for 2 h. Cells were lysed and viable bacteria recovered. Asterisks (*) represent statistical comparisons between alcohol treatment and (Non-Alcohol) control determined by one-way ANOVA. Bars represent average CFU with SEM. **, p ≤ .01; ***, p ≤ .001; ****, p ≤ 0.0001.

    Article Snippet: Cell monolayers were grown to confluency on 0.4 micron ThinCert Transwells (Greiner Bio-One, Germany) with DMEM F12 medium (Gibco, Life Technologies) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 0.1 mM non-essential amino acids, 1.5 g/l sodium bicarbonate, 50 U/ml penicillin, and 50 mg/ml streptomycin.

    Techniques: Cell Culture

    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Article Snippet: RNA interference studies SH-SY5Y cells expressing PrPC were seeded into T80 flasks at 70% confluency and incubated with 500 pmol of a 2 µM Smartpool siRNA solution against glypican-1 or a control Smartpool reagent (Dharmacon Inc., Chicago, U.S.A.) complexed with DharmaFECT-1 transfection regent (Dharmacon Inc.) in serum-free medium.

    Techniques: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Ift88 expedites cell migration in previously unciliated MDCK cells. (A) Ift88-i1 cells were grown to confluency for 2 days (unciliated stage, compare Fig 3 ) and subjected to wounding. Cells depleted of Ift88 by inducible shRNA (+Tet) migrate more slowly compared with non-induced control cells (-Tet). The leading edge is shown after 2h and 6h. Scale bars: 100μm. (B) Quantification reveals no significant reduction of migration speed for a control cell line: -Tet: 157.1 ±12.8 μm 2 /minute vs. +Tet: 144.8 ±10.6 μm 2 /minute (n.s.; p = 0.5). Migration speed is reduced in two independent cell lines after induced depletion of Ift88. Ift88-i1: -Tet: 133.9 ±7.0 μm 2 /minute vs. +Tet: 50.4 ±2.5 μm 2 /minute, p

    Journal: PLoS ONE

    Article Title: A Cilia Independent Role of Ift88/Polaris during Cell Migration

    doi: 10.1371/journal.pone.0140378

    Figure Lengend Snippet: Ift88 expedites cell migration in previously unciliated MDCK cells. (A) Ift88-i1 cells were grown to confluency for 2 days (unciliated stage, compare Fig 3 ) and subjected to wounding. Cells depleted of Ift88 by inducible shRNA (+Tet) migrate more slowly compared with non-induced control cells (-Tet). The leading edge is shown after 2h and 6h. Scale bars: 100μm. (B) Quantification reveals no significant reduction of migration speed for a control cell line: -Tet: 157.1 ±12.8 μm 2 /minute vs. +Tet: 144.8 ±10.6 μm 2 /minute (n.s.; p = 0.5). Migration speed is reduced in two independent cell lines after induced depletion of Ift88. Ift88-i1: -Tet: 133.9 ±7.0 μm 2 /minute vs. +Tet: 50.4 ±2.5 μm 2 /minute, p

    Article Snippet: Wound healing and migration of subconfluent cells For wound healing assays, MDCK cells were plated and grown to confluency on Ibidi μ-dishes (Ibidi, GmbH, Munich, Germany) coated with collagen A (Biochrom AG, Berlin, Germany).

    Techniques: Migration, shRNA

    Ift88 expedites cell migration in ciliated MDCK cells. (A) Ift88-i1 cells were grown to confluency for 7–8 days (ciliated stage) and subjected to wounding. Cells depleted of Ift88 by inducible shRNA (+Tet) migrate slower compared with non-induced control cells (-Tet). The leading edge is shown after 2h and 6h. Scale bars: 100μm. (B) Quantification of migration speed in different cell lines expressing different tetracycline inducible shRNAs. No significant (n.s.) reduction in migration speed is observed in cells expressing unspecific shRNA (control; -Tet: 111.5 ±5.8 μm 2 /minute vs. 105.3 ±10.2 μm 2 /minute, p = 0.46, n = 4). Two independent cell lines expressing different shRNAs against Ift88 show significantly reduced migration speed: Ift88-i1: -Tet: 111.0 ±4.7 μm 2 /minute vs. +Tet: 64.6 ±10.3 μm 2 /minute, p

    Journal: PLoS ONE

    Article Title: A Cilia Independent Role of Ift88/Polaris during Cell Migration

    doi: 10.1371/journal.pone.0140378

    Figure Lengend Snippet: Ift88 expedites cell migration in ciliated MDCK cells. (A) Ift88-i1 cells were grown to confluency for 7–8 days (ciliated stage) and subjected to wounding. Cells depleted of Ift88 by inducible shRNA (+Tet) migrate slower compared with non-induced control cells (-Tet). The leading edge is shown after 2h and 6h. Scale bars: 100μm. (B) Quantification of migration speed in different cell lines expressing different tetracycline inducible shRNAs. No significant (n.s.) reduction in migration speed is observed in cells expressing unspecific shRNA (control; -Tet: 111.5 ±5.8 μm 2 /minute vs. 105.3 ±10.2 μm 2 /minute, p = 0.46, n = 4). Two independent cell lines expressing different shRNAs against Ift88 show significantly reduced migration speed: Ift88-i1: -Tet: 111.0 ±4.7 μm 2 /minute vs. +Tet: 64.6 ±10.3 μm 2 /minute, p

    Article Snippet: Wound healing and migration of subconfluent cells For wound healing assays, MDCK cells were plated and grown to confluency on Ibidi μ-dishes (Ibidi, GmbH, Munich, Germany) coated with collagen A (Biochrom AG, Berlin, Germany).

    Techniques: Migration, shRNA, Expressing

    Lipid vacuole formation and gene expression for adipogenic differentiation. AdMSCs were allowed to reach confluency and were then exposed to 0.5% CSE at the moment of induction of adipogenic differentiation with supplemented medium and for 24 h after induction. ( A ) After 21 d in culture, the cells were stained with Oil Red O to detect lipid vacuoles (10× magnification). ( B ) The Oil Red O solution was eluted and the optical density was measured, indicating there were no significant differences in lipid vacuole formation after acute CSE exposure. ( C ) RNA was extracted from the cells and the gene expression of early ( PPARγ ), late ( LEP ), and abundant ( ADIPOQ ) markers was analysed with qPCR, revealing no significant differences after 3 d.

    Journal: Scientific Reports

    Article Title: Acute stimulation of mesenchymal stem cells with cigarette smoke extract affects their migration, differentiation, and paracrine potential

    doi: 10.1038/srep22957

    Figure Lengend Snippet: Lipid vacuole formation and gene expression for adipogenic differentiation. AdMSCs were allowed to reach confluency and were then exposed to 0.5% CSE at the moment of induction of adipogenic differentiation with supplemented medium and for 24 h after induction. ( A ) After 21 d in culture, the cells were stained with Oil Red O to detect lipid vacuoles (10× magnification). ( B ) The Oil Red O solution was eluted and the optical density was measured, indicating there were no significant differences in lipid vacuole formation after acute CSE exposure. ( C ) RNA was extracted from the cells and the gene expression of early ( PPARγ ), late ( LEP ), and abundant ( ADIPOQ ) markers was analysed with qPCR, revealing no significant differences after 3 d.

    Article Snippet: Adipogenic differentiation Cells were seeded at a density of 21,000/cm2 and left to attach for 2–3 d until they reached confluency, upon which time they were stimulated with CSE and adipogenic induction medium (Lonza).

    Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction

    Aβ peptides inhibit migration of hP1-CL cells. Cell migration was measured as cell movement on glass cover slips in polystyrene dishes at 37 °C and 5% CO 2 . A PDMS barrier to cell growth was created on the cover slip. When cells reached confluency, the barrier was peeled off. Images of the resulting, initially sharp confluent cell boundary were captured at 0.003 Hz over 5 hours. Top panel shows initial (t = 0 min, black dotted lines) and final time points (t = 240 min, red dotted lines) of cell migration. hP1-CL cell migration was blocked by 5 µM D-GsMTx4, as well as 10 pM concentrations of L and D Aβ(1-40). Bottom left panel summarizes the mean cell migration distances as a function of time. Bottom right panel is the mean migration rate of hP1-CL cells in presence and absence of 5 µM D-GsMTx4, and of 10 pM of the L or D forms of Aβ(1-40). Three independent experiments were averaged (SEM).

    Journal: Scientific Reports

    Article Title: Enantiomeric Aβ peptides inhibit the fluid shear stress response of PIEZO1

    doi: 10.1038/s41598-018-32572-2

    Figure Lengend Snippet: Aβ peptides inhibit migration of hP1-CL cells. Cell migration was measured as cell movement on glass cover slips in polystyrene dishes at 37 °C and 5% CO 2 . A PDMS barrier to cell growth was created on the cover slip. When cells reached confluency, the barrier was peeled off. Images of the resulting, initially sharp confluent cell boundary were captured at 0.003 Hz over 5 hours. Top panel shows initial (t = 0 min, black dotted lines) and final time points (t = 240 min, red dotted lines) of cell migration. hP1-CL cell migration was blocked by 5 µM D-GsMTx4, as well as 10 pM concentrations of L and D Aβ(1-40). Bottom left panel summarizes the mean cell migration distances as a function of time. Bottom right panel is the mean migration rate of hP1-CL cells in presence and absence of 5 µM D-GsMTx4, and of 10 pM of the L or D forms of Aβ(1-40). Three independent experiments were averaged (SEM).

    Article Snippet: To generate viral particles, AmphoPak cells in 30 mm dishes were grown to 25% confluency and transfected with DNA (1 µg per dish) using Mirus transfection reagent according to manufacturer’s recommendation.

    Techniques: Migration