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  • 99
    Millipore flow cytometry flow cytometric analyses
    <t>Flow-cytometric</t> analysis of surface markers (CD11c, CD11b, and MHC class II) during generation of immature DCs from bone marrow cells in the presence of 10 ng/mL of granulocyte macrophage colony-stimulating factor on days 0, 3, and 7 of culture. Note: Numbers within dot plots represent percentages within the quadrant. Abbreviations: DCs, dendritic cells; MHC, major histocompatibility complex.
    Flow Cytometry Flow Cytometric Analyses, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 645 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher flow cytometry flow cytometric reference beads
    <t>Flow-cytometric</t> analysis of surface markers (CD11c, CD11b, and MHC class II) during generation of immature DCs from bone marrow cells in the presence of 10 ng/mL of granulocyte macrophage colony-stimulating factor on days 0, 3, and 7 of culture. Note: Numbers within dot plots represent percentages within the quadrant. Abbreviations: DCs, dendritic cells; MHC, major histocompatibility complex.
    Flow Cytometry Flow Cytometric Reference Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore flow cytometry flow cytometry
    Generation of A549-derived cell-lines. A . Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences ( 5p and 3p ) are indicated. B . Histograms for red fluorescence quantified by flow <t>cytometry</t> of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b ( A549/146b ), or its variant shown in A ( A549/v146b ), or neither ( A549/vec ). C . Comparison of miR-146b-5p and -3p levels ( 5p and 3p ), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D . Standard curves showing the relationship between quantification cycle ( C q ) value and concentration of RNA in miR-146b-5p and -3p ( 5p and 3p ) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log 2 scale is used for the X axis. E . Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.
    Flow Cytometry Flow Cytometry, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3641 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Miltenyi Biotec flow cytometry cytometric analysis
    Generation of A549-derived cell-lines. A . Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences ( 5p and 3p ) are indicated. B . Histograms for red fluorescence quantified by flow <t>cytometry</t> of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b ( A549/146b ), or its variant shown in A ( A549/v146b ), or neither ( A549/vec ). C . Comparison of miR-146b-5p and -3p levels ( 5p and 3p ), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D . Standard curves showing the relationship between quantification cycle ( C q ) value and concentration of RNA in miR-146b-5p and -3p ( 5p and 3p ) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log 2 scale is used for the X axis. E . Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.
    Flow Cytometry Cytometric Analysis, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson flow cytometry a flow cytometric assay
    Generation of A549-derived cell-lines. A . Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences ( 5p and 3p ) are indicated. B . Histograms for red fluorescence quantified by flow <t>cytometry</t> of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b ( A549/146b ), or its variant shown in A ( A549/v146b ), or neither ( A549/vec ). C . Comparison of miR-146b-5p and -3p levels ( 5p and 3p ), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D . Standard curves showing the relationship between quantification cycle ( C q ) value and concentration of RNA in miR-146b-5p and -3p ( 5p and 3p ) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log 2 scale is used for the X axis. E . Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.
    Flow Cytometry A Flow Cytometric Assay, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson flow cytometry flow cytometry
    The pPAC7 vector . A) Schematic map of the pPAC7 vector which contains the EGFP gene cassette (EGFP), the blasticidin deaminase gene cassette (Bsr), the EBNA-1 gene cassette (EBNA-1), and the Epstein Barr virus origin of replication ( ori P). The pUC-link fragment is removed by BamH I or Not I digestion during the cloning process and replaced by large genomic DNA fragments allowing positive selection for recombinant clones on sucrose containing media due to the Sac BII gene present on the vector. The kanamycin resistance gene (Kan(R)) is a bacterial selection marker. The Not I restriction sites are used when verifying the different constructs. B) SK-BR-3 cells were transfected with pPAC7 and grown in the presence of blasticidin using the concentrations indicated. The percent of EGFP positive cells, from all viable cells, were measured by flow <t>cytometry</t> after 2, 4 and 6 days. The experiments were performed in triplicates with standard deviations shown. C) SK-BR-3 cells transfected with pPAC7 or pEGFP-C1 were sorted by flow cytometry and grown in non-selective medium. The percentage and total number of EGFP expressing cells was measured by flow cytometry 4, 11 and 15 days after cell sorting. The experiments were performed in triplicates with standard deviations indicated. D) The fluorescent microscopy pictures show the SK-BR-3 cells transfected with pEGFP-C1 and pPAC7 15 days after cell sorting. The shutter time is 1306 ms for pEGFP-C1 transfected and 390 ms for pPAC7 transfected cells.
    Flow Cytometry Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1984 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Flow-cytometric analysis of surface markers (CD11c, CD11b, and MHC class II) during generation of immature DCs from bone marrow cells in the presence of 10 ng/mL of granulocyte macrophage colony-stimulating factor on days 0, 3, and 7 of culture. Note: Numbers within dot plots represent percentages within the quadrant. Abbreviations: DCs, dendritic cells; MHC, major histocompatibility complex.

    Journal: Drug Design, Development and Therapy

    Article Title: Effects of histamine and its antagonists on murine T-cells and bone marrow-derived dendritic cells

    doi: 10.2147/DDDT.S89792

    Figure Lengend Snippet: Flow-cytometric analysis of surface markers (CD11c, CD11b, and MHC class II) during generation of immature DCs from bone marrow cells in the presence of 10 ng/mL of granulocyte macrophage colony-stimulating factor on days 0, 3, and 7 of culture. Note: Numbers within dot plots represent percentages within the quadrant. Abbreviations: DCs, dendritic cells; MHC, major histocompatibility complex.

    Article Snippet: Flow-cytometric analysis of murine spleen cells For in vitro stimulation, 2×106 spleen cells were mixed with 10 ng/mL of 4beta-phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 1 µg/mL of ionomycin (Sigma-Aldrich) and incubated in a 24-well plate at 37°C and 5% CO2 .

    Techniques: Flow Cytometry

    Fraction of cells with decreased mitochondrial potential and increased [Ca 2+ ] i after Hemidesmus treatment. Alterations in mitochondrial membrane permeability in absence (A) and presence (B) of inhibitors following treatment with Hemidesmus 0.93 mg/mL. Fraction of cells with increased [Ca 2+ ] i following 3 or 24 h culture in the absence or presence of Hemidesmus (0.93 mg/mL) (C), flow cytometric analysis of [Ca 2+ ] i following 24 h culture in the absence or presence of Hemidesmus (0.93 mg/mL) (D). M1 and M2 indicate the two different populations characterized by a different mean fluorescence intensity values. Data are means ± SEM of four independent experiments.

    Journal: PLoS ONE

    Article Title: Mitochondrial Pathway Mediates the Antileukemic Effects of Hemidesmus Indicus, a Promising Botanical Drug

    doi: 10.1371/journal.pone.0021544

    Figure Lengend Snippet: Fraction of cells with decreased mitochondrial potential and increased [Ca 2+ ] i after Hemidesmus treatment. Alterations in mitochondrial membrane permeability in absence (A) and presence (B) of inhibitors following treatment with Hemidesmus 0.93 mg/mL. Fraction of cells with increased [Ca 2+ ] i following 3 or 24 h culture in the absence or presence of Hemidesmus (0.93 mg/mL) (C), flow cytometric analysis of [Ca 2+ ] i following 24 h culture in the absence or presence of Hemidesmus (0.93 mg/mL) (D). M1 and M2 indicate the two different populations characterized by a different mean fluorescence intensity values. Data are means ± SEM of four independent experiments.

    Article Snippet: Flow cytometry All flow cytometric procedures were performed with a Guava EasyCyte Mini flow cytometer (Guava Technologies-Millipore, Hayward, CA, USA).

    Techniques: Permeability, Flow Cytometry, Fluorescence

    ATM-depleted fibroblasts progressively accumulate ROS and oxidative protein damage. ( A ) Representative Western blot analysis on ATM-depleted fibroblasts showing upregulation of proteins involved in the antioxidant response. TIG1 cells were transfected with either a control siRNA (siCtrl) or an ATM-targeting siRNA (siATM#1, siATM #2). Actin was used as loading control. Densitometric quantification of the indicated proteins is reported at the bottom of the gel ( N = 2). ( B ) Western blot analysis on a representative time course depletion of ATM. TIG1 fibroblasts were treated as indicated and ATM expression was monitored in nuclear cell extracts. Lamin A/C was used as loading control. ( C ) Quantification of ROS content using flow-cytometry in siATM-treated fibroblasts. TIG1 fibroblasts were treated as indicated. H 2 O 2 (25 μM, 30 min) was used as a positive control for induction of ROS. ATM inhibitors (Ku-55933 – ATMi #1 and Ku-60019 – ATMi #2, both 10 μM) were provided fresh every 24 h for a total of 72 h ( N = 3). ( D ) Quantification of mitochondrial membrane potential (ΔΨm) in siATM-treated fibroblasts using flow-cytometry. Cells were treated as in panel C. Staurosporine (2 μM, 2 h) was used as a positive control for mitochondrial membrane depolarisation ( N = 3). ( E ) Detection of protein carbonylation in nuclear extracts obtained from fibroblasts depleted of ATM for the indicated time. Carbonylated proteins were detected by derivatisation with dinitrophenylhydrazone (DNP) followed by Western blot using an anti-DNP antibody. Equal amounts of cell extract were loaded in each lane. H 2 O 2 (1 mM, 30 minutes) was used as a positive control for induction of protein carbonylation. ( F ) Quantification of protein carbonylation in ATM-depleted fibroblasts; the histogram displays densitometric data from the analysis reported in panel E ( N = 6). Results are expressed as mean ± SD from the indicated number ( N ) of independent experiments: * P

    Journal: Nucleic Acids Research

    Article Title: Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells

    doi: 10.1093/nar/gkx635

    Figure Lengend Snippet: ATM-depleted fibroblasts progressively accumulate ROS and oxidative protein damage. ( A ) Representative Western blot analysis on ATM-depleted fibroblasts showing upregulation of proteins involved in the antioxidant response. TIG1 cells were transfected with either a control siRNA (siCtrl) or an ATM-targeting siRNA (siATM#1, siATM #2). Actin was used as loading control. Densitometric quantification of the indicated proteins is reported at the bottom of the gel ( N = 2). ( B ) Western blot analysis on a representative time course depletion of ATM. TIG1 fibroblasts were treated as indicated and ATM expression was monitored in nuclear cell extracts. Lamin A/C was used as loading control. ( C ) Quantification of ROS content using flow-cytometry in siATM-treated fibroblasts. TIG1 fibroblasts were treated as indicated. H 2 O 2 (25 μM, 30 min) was used as a positive control for induction of ROS. ATM inhibitors (Ku-55933 – ATMi #1 and Ku-60019 – ATMi #2, both 10 μM) were provided fresh every 24 h for a total of 72 h ( N = 3). ( D ) Quantification of mitochondrial membrane potential (ΔΨm) in siATM-treated fibroblasts using flow-cytometry. Cells were treated as in panel C. Staurosporine (2 μM, 2 h) was used as a positive control for mitochondrial membrane depolarisation ( N = 3). ( E ) Detection of protein carbonylation in nuclear extracts obtained from fibroblasts depleted of ATM for the indicated time. Carbonylated proteins were detected by derivatisation with dinitrophenylhydrazone (DNP) followed by Western blot using an anti-DNP antibody. Equal amounts of cell extract were loaded in each lane. H 2 O 2 (1 mM, 30 minutes) was used as a positive control for induction of protein carbonylation. ( F ) Quantification of protein carbonylation in ATM-depleted fibroblasts; the histogram displays densitometric data from the analysis reported in panel E ( N = 6). Results are expressed as mean ± SD from the indicated number ( N ) of independent experiments: * P

    Article Snippet: Flow cytometry Determination of intracellular ROS content was carried out by staining with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Sigma).

    Techniques: Western Blot, Transfection, Expressing, Flow Cytometry, Cytometry, Positive Control

    Generation of A549-derived cell-lines. A . Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences ( 5p and 3p ) are indicated. B . Histograms for red fluorescence quantified by flow cytometry of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b ( A549/146b ), or its variant shown in A ( A549/v146b ), or neither ( A549/vec ). C . Comparison of miR-146b-5p and -3p levels ( 5p and 3p ), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D . Standard curves showing the relationship between quantification cycle ( C q ) value and concentration of RNA in miR-146b-5p and -3p ( 5p and 3p ) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log 2 scale is used for the X axis. E . Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.

    Journal: PLoS ONE

    Article Title: Overexpression of the Lung Cancer-Prognostic miR-146b MicroRNAs Has a Minimal and Negative Effect on the Malignant Phenotype of A549 Lung Cancer Cells

    doi: 10.1371/journal.pone.0022379

    Figure Lengend Snippet: Generation of A549-derived cell-lines. A . Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences ( 5p and 3p ) are indicated. B . Histograms for red fluorescence quantified by flow cytometry of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b ( A549/146b ), or its variant shown in A ( A549/v146b ), or neither ( A549/vec ). C . Comparison of miR-146b-5p and -3p levels ( 5p and 3p ), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D . Standard curves showing the relationship between quantification cycle ( C q ) value and concentration of RNA in miR-146b-5p and -3p ( 5p and 3p ) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log 2 scale is used for the X axis. E . Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.

    Article Snippet: Flow cytometry Flow cytometry of freshly-harvested cells stained with 1 µg/ml of the 7 -amino-actinomycin D viability dye (Sigma®, St. Louis, MO) was performed on a FACSCalibur machine (BD Biosciences®, San Jose, CA).

    Techniques: Derivative Assay, Variant Assay, Fluorescence, Flow Cytometry, Cytometry, Stable Transfection, Transduction, Construct, Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Concentration Assay

    Existence of stem-like side population cells in the human retinoblastoma WERI-Rb1 cell line. The cells were stained with Hoechst 33,342 and sorted with flow cytometry.  A : A small population of cells (0.075±0.017%) was identified.  B : Addition of 50 μM verapamil markedly reduced the staining of the SP cells. (n=3; x-axis, Hoechst red fluorescence intensity; y-axis, Hoechst blue fluorescence intensity).

    Journal: Molecular Vision

    Article Title: Characterization and retinal neuron differentiation of WERI-Rb1 cancer stem cells

    doi:

    Figure Lengend Snippet: Existence of stem-like side population cells in the human retinoblastoma WERI-Rb1 cell line. The cells were stained with Hoechst 33,342 and sorted with flow cytometry. A : A small population of cells (0.075±0.017%) was identified. B : Addition of 50 μM verapamil markedly reduced the staining of the SP cells. (n=3; x-axis, Hoechst red fluorescence intensity; y-axis, Hoechst blue fluorescence intensity).

    Article Snippet: CD133 staining for flow cytometry A sample of 1×107 cells in suspension was collected, and the cells were resuspended in PBS with 0.5% BSA (BSA; Sigma-Aldrich) and 2 mM EDTA (Sigma-Aldrich).

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence

    (a) Confocal fluorescence microscopy (CFM) images of KB and A549 cells after treated with different GNPs. All GNPs were labeled with TA-FITC which had green fluorescence. The scale bar is 50 μm. Flow cytometry assay to monitor the binding of GNPs

    Journal: Biomaterials

    Article Title: Enhancement of cell recognition in vitro by dual-ligand cancer targeting gold naoparticles

    doi: 10.1016/j.biomaterials.2010.12.031

    Figure Lengend Snippet: (a) Confocal fluorescence microscopy (CFM) images of KB and A549 cells after treated with different GNPs. All GNPs were labeled with TA-FITC which had green fluorescence. The scale bar is 50 μm. Flow cytometry assay to monitor the binding of GNPs

    Article Snippet: Flow cytometry analyses were performed on a Guava EasyCyte Mini flow cytometry system (Millipore, Billerica, MA).

    Techniques: Fluorescence, Microscopy, Labeling, Flow Cytometry, Cytometry, Binding Assay

    Frequency of monocytes subsets. (A) Representative gates of flow cytometry for classification of subsets. (B) Frequency of each monocyte subsets in normal-weight (NW; n = 9) and childhood obesity (CO; n = 11). Graphs shown median with interquartile range. Significant differences (p

    Journal: PLoS ONE

    Article Title: Chronic Low-Grade Inflammation in Childhood Obesity Is Associated with Decreased IL-10 Expression by Monocyte Subsets

    doi: 10.1371/journal.pone.0168610

    Figure Lengend Snippet: Frequency of monocytes subsets. (A) Representative gates of flow cytometry for classification of subsets. (B) Frequency of each monocyte subsets in normal-weight (NW; n = 9) and childhood obesity (CO; n = 11). Graphs shown median with interquartile range. Significant differences (p

    Article Snippet: Flow cytometry analysis of peripheral blood Peripheral blood from patients was incubated for 4 hours in 5% CO2 at 37°C with Brefeldin-A (SIGMA-USA) (10μg/ml).

    Techniques: Flow Cytometry, Cytometry

    Purity verification of separated CD4 + T cells by flow cytometry. (a) showed the isotype control performed by Simultest IgG2a/IgG1. (b) represents double staining by anti-human CD3 and CD4 antibody. (c) represents anti-human antibody CD4 staining. These results showed that the purity of CD4 + from PBMC can reach a percent of more than 95%.

    Journal: Mediators of Inflammation

    Article Title: Clinical Significance of IL-23 Regulating IL-17A and/or IL-17F Positive Th17 Cells in Chronic Periodontitis

    doi: 10.1155/2014/627959

    Figure Lengend Snippet: Purity verification of separated CD4 + T cells by flow cytometry. (a) showed the isotype control performed by Simultest IgG2a/IgG1. (b) represents double staining by anti-human CD3 and CD4 antibody. (c) represents anti-human antibody CD4 staining. These results showed that the purity of CD4 + from PBMC can reach a percent of more than 95%.

    Article Snippet: Flow Cytometry Detection of IL-17A+ and/or IL-17F+ Th17 Cells After stimulation with rhIL-23 for 24 hours, cells were cultured in the presence of 50 ng/mL phorbol 12-myristate 13-acetate (P1585, Sigma St. Louis, MO, USA) and 500 ng/mL ionomycin (I0634, Sigma, USA) and with the protein transport inhibitor BD Golgistop 0.7 μ L/mL (Cat. number 554714, BD Bioscience) to prevent the cytokine secretion and allow cytokine accumulation inside the cell.

    Techniques: Flow Cytometry, Cytometry, Double Staining, Staining

    Inhibition of Axl restores ponatinib sensitivity in the K562-R and K562 DOX-R cell lines while AXL knockdown increases ponatinib sensitivity (A, B) Viability as assessed by Flow cytometry (AnnexinV-PE/7AAD double negative). The presence of the Axl inhibitor R428 (1 μM) or BMS777607 (12.5 μM) induced cell death significantly in (A) K562-R and (B) K562 DOX-R when co-treated with 200 nM ponatinib compared to their corresponding control lines. (C, D) Cell surface Axl expression was measured by flow cytometry in the AXL or scramble control shRNA transduced resistant cell lines. Compared to scramble control, AXL shRNA transduced cells demonstrated a reduction in Axl expression on the cell surface. (E, F) AXL knockdown in the K562 and K562 DOX ponatinib resistant cell lines demonstrated re-sensitisation to 10 nM ponatinib. Error bars represent SD, n≥3. * p

    Journal: Oncotarget

    Article Title: Modelling ponatinib resistance in tyrosine kinase inhibitor-naïve and dasatinib resistant BCR-ABL1+ cell lines

    doi: 10.18632/oncotarget.26187

    Figure Lengend Snippet: Inhibition of Axl restores ponatinib sensitivity in the K562-R and K562 DOX-R cell lines while AXL knockdown increases ponatinib sensitivity (A, B) Viability as assessed by Flow cytometry (AnnexinV-PE/7AAD double negative). The presence of the Axl inhibitor R428 (1 μM) or BMS777607 (12.5 μM) induced cell death significantly in (A) K562-R and (B) K562 DOX-R when co-treated with 200 nM ponatinib compared to their corresponding control lines. (C, D) Cell surface Axl expression was measured by flow cytometry in the AXL or scramble control shRNA transduced resistant cell lines. Compared to scramble control, AXL shRNA transduced cells demonstrated a reduction in Axl expression on the cell surface. (E, F) AXL knockdown in the K562 and K562 DOX ponatinib resistant cell lines demonstrated re-sensitisation to 10 nM ponatinib. Error bars represent SD, n≥3. * p

    Article Snippet: Immunophenotyping by Flow Cytometry: measurement of protein cell surface expression 5 × 105 cells were incubated with Hanks Balanced Salt Solution (HBSS) (JRH Biosciences, Australia) supplemented with 10mM Hepes (Sigma-Aldrich) and with corresponding isotype controls: IgG2aPE/IgG2bPE (Dako, Australia), IgG1PE (BD Biosciences, Australia); or PE-conjugated anti-hBCRP1 (ABCG2-PE) (R & D Systems, Australia), PE-conjugated anti-CD243 (ABCB1-PE) (Beckman Coulter, Australia), PE-conjugated anti-hAxl (R & D Systems, Australia) or PE-conjugated anti-hCD44 (BD Biosciences, Australia) antibodies for 40 minutes on ice.

    Techniques: Inhibition, Flow Cytometry, Cytometry, Expressing, shRNA

    Resistant cell lines K562-R and K562 DOX-R demonstrate increased adhesion compared to their corresponding control lines (A) Adherent cells were observed in both resistant cell lines, but not in their respective control lines, by light microscopy with 20X magnification. (B) Cells from resistant and control cultures were analysed by flow cytometry for expression of the adhesion marker CD44 (CD44-PE green; unstained grey; IgG2b PE isotype control red).

    Journal: Oncotarget

    Article Title: Modelling ponatinib resistance in tyrosine kinase inhibitor-naïve and dasatinib resistant BCR-ABL1+ cell lines

    doi: 10.18632/oncotarget.26187

    Figure Lengend Snippet: Resistant cell lines K562-R and K562 DOX-R demonstrate increased adhesion compared to their corresponding control lines (A) Adherent cells were observed in both resistant cell lines, but not in their respective control lines, by light microscopy with 20X magnification. (B) Cells from resistant and control cultures were analysed by flow cytometry for expression of the adhesion marker CD44 (CD44-PE green; unstained grey; IgG2b PE isotype control red).

    Article Snippet: Immunophenotyping by Flow Cytometry: measurement of protein cell surface expression 5 × 105 cells were incubated with Hanks Balanced Salt Solution (HBSS) (JRH Biosciences, Australia) supplemented with 10mM Hepes (Sigma-Aldrich) and with corresponding isotype controls: IgG2aPE/IgG2bPE (Dako, Australia), IgG1PE (BD Biosciences, Australia); or PE-conjugated anti-hBCRP1 (ABCG2-PE) (R & D Systems, Australia), PE-conjugated anti-CD243 (ABCB1-PE) (Beckman Coulter, Australia), PE-conjugated anti-hAxl (R & D Systems, Australia) or PE-conjugated anti-hCD44 (BD Biosciences, Australia) antibodies for 40 minutes on ice.

    Techniques: Light Microscopy, Flow Cytometry, Cytometry, Expressing, Marker

    Very delayed rPostC reverses post-stroke peripheral immunosuppression. Flow cytometry analysis from blood samples was performed 7 days after stroke induction in mice that had received rPostC 120 h after stroke or in control mice that had not received rPostC (non-rPostC). Sham animals underwent surgical procedure for transient focal cerebral ischemia without inserting of the filament into the left middle cerebral artery. A quantitative analysis of leukocytes (A) , neutrophils (B) , B-lymphocytes (C) , and T-lymphocytes (D) was performed as described in the materials and methods section. ∗ Significantly different from non-rPostC, ( p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Very Delayed Remote Ischemic Post-conditioning Induces Sustained Neurological Recovery by Mechanisms Involving Enhanced Angioneurogenesis and Peripheral Immunosuppression Reversal

    doi: 10.3389/fncel.2018.00383

    Figure Lengend Snippet: Very delayed rPostC reverses post-stroke peripheral immunosuppression. Flow cytometry analysis from blood samples was performed 7 days after stroke induction in mice that had received rPostC 120 h after stroke or in control mice that had not received rPostC (non-rPostC). Sham animals underwent surgical procedure for transient focal cerebral ischemia without inserting of the filament into the left middle cerebral artery. A quantitative analysis of leukocytes (A) , neutrophils (B) , B-lymphocytes (C) , and T-lymphocytes (D) was performed as described in the materials and methods section. ∗ Significantly different from non-rPostC, ( p

    Article Snippet: For flow cytometry measurements of brain tissue samples, ischemic left hemispheres were mechanically homogenized in a buffer of collagenase type XI (125 U/ml), hyaluronidase (60 U/ml) and collagenase (450 U/ml) in Ca2+ /Mg2+ supplemented PBS (Sigma-Aldrich, Germany).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay

    T-cell intrinsic TLR2 signals are not required for effector T-cell or Treg functions in vivo . (A) CD4 + T cells from the spleen, MLN and colonic LPL were analyzed for Foxp3 expression by flow cytometry. Data represents group means (±SEM) from two pooled independent experiments ( n =8–10 mice per group). (B) NaYve CD45RB hi (RB hi ), memory CD25 − CD45RB lo (CD25 − ) and Treg CD25 + CD45RB lo (CD25 + ) CD4 + T cells were sorted from the MLN and LPL of B6 WT mice ( n =5) and analyzed for TLR2 gene expression by Q-PCR. Data represent means±SEM normalized relative to HPRT. (C) B6.RAG −/− mice were reconstituted with either 4×10 5 WT or TLR2 −/− CD4 + CD45RB hi T cells alone (colitis “induction”) or concurrently with 2×10 5 WT or TLR2 −/− CD4 + CD25 + Treg (colitis “protection”). After 8–10 wk colonic pathology was assessed histologically. Each symbol represents a single animal and the data represents pooled results of two independent experiments ( n =10–14 mice per group). Horizontal lines represent group means and differences were assessed using the Mann–Whitney U -test; ns, not significant.

    Journal: European Journal of Immunology

    Article Title: TLR2-independent induction and regulation of chronic intestinal inflammation

    doi: 10.1002/eji.200939669

    Figure Lengend Snippet: T-cell intrinsic TLR2 signals are not required for effector T-cell or Treg functions in vivo . (A) CD4 + T cells from the spleen, MLN and colonic LPL were analyzed for Foxp3 expression by flow cytometry. Data represents group means (±SEM) from two pooled independent experiments ( n =8–10 mice per group). (B) NaYve CD45RB hi (RB hi ), memory CD25 − CD45RB lo (CD25 − ) and Treg CD25 + CD45RB lo (CD25 + ) CD4 + T cells were sorted from the MLN and LPL of B6 WT mice ( n =5) and analyzed for TLR2 gene expression by Q-PCR. Data represent means±SEM normalized relative to HPRT. (C) B6.RAG −/− mice were reconstituted with either 4×10 5 WT or TLR2 −/− CD4 + CD45RB hi T cells alone (colitis “induction”) or concurrently with 2×10 5 WT or TLR2 −/− CD4 + CD25 + Treg (colitis “protection”). After 8–10 wk colonic pathology was assessed histologically. Each symbol represents a single animal and the data represents pooled results of two independent experiments ( n =10–14 mice per group). Horizontal lines represent group means and differences were assessed using the Mann–Whitney U -test; ns, not significant.

    Article Snippet: Flow cytometry Aliquots of ∼1×106 spleen cells in HBSS/0.1% BSA (Sigma Aldrich) were incubated for 30 min with FcR block (eBioscience) at 4°C, and surface staining steps performed for 20 min at 4°C with a panel of monoclonal antibodies as follows: biotinylated anti-DX5, biotinylated anti-Gr1, PE anti-CD11c and APC anti-CD11b (all obtained from BD Bioscience).

    Techniques: In Vivo, Expressing, Flow Cytometry, Cytometry, Mouse Assay, Polymerase Chain Reaction, MANN-WHITNEY

    Evaluation of AP contribution to adipogenesis in mgWAT. a , b Representative flow cytometry plots of CD31−/CD45− (Lin−), CD34+, CD29+, Sca-1+, CD24± populations from the stromal vascular fraction (SVF) of fixed murine inguinal/mgWAT at involution day 1 ( a , left panel) or CD45−, CD34+, CD90+ cells within the SVF of human breast tissue ( b , left panel). Representative images of unpurified cells in total SVF or FACS purified adipocyte precursors (APs) from mice ( a , right panel) or humans ( b , right panel) after culturing in adipogenic media for 8 days ( a ) or 13 days ( b ) and staining with Oil Red O. n = 3 biological repeats. c Schematic summarizing stages of adipogenesis. d Experimental strategy to measure EdU incorporation in adipocyte precursors by FACS (as in e and f ), and in mature adipocytes (as in g , h ). e Representative flow cytometry plots of EdU signal in APs (as defined in a ) in virgin mice and at involution days 1 and 3. f The average percentage of EdU+ APs identified with flow cytometry as in e . n = 4 mice per time point, P = 0.51 for virgin vs. involution day 1 data. g, h Representative image ( g ) and quantification ( h ) of EdU immunofluorescence in Adi p oq-Cre; mT/mG mouse injected with EdU according to the timeline in d . Note EdU+ (arrow) and EdU− nuclei (arrowhead) in mGFP+ adipocytes. White box indicates insets. n = 200–800 nuclei in each of four mice per group, P = 0.84. i Experimental strategy to inhibit adipogenesis during involution using GW9662, a pharmacological antagonist of PPARγ. j , k Representative image ( j ) and quantification ( k ) of MGs from GW9662- and vehicle-treated mice, stained with a perilipin antibody and DAPI. N = 8–17 fields in each of five mice per treatment group, P = 0.09. Scale bar is 50 μm in g and j . Data are mean ± SEM. Statistics were performed using a one-way ANOVA with Tukey’s multiple comparisons test ( f ) and a two-tailed unpaired t -test ( h , k ). **** P

    Journal: Nature Communications

    Article Title: Adipocyte hypertrophy and lipid dynamics underlie mammary gland remodeling after lactation

    doi: 10.1038/s41467-018-05911-0

    Figure Lengend Snippet: Evaluation of AP contribution to adipogenesis in mgWAT. a , b Representative flow cytometry plots of CD31−/CD45− (Lin−), CD34+, CD29+, Sca-1+, CD24± populations from the stromal vascular fraction (SVF) of fixed murine inguinal/mgWAT at involution day 1 ( a , left panel) or CD45−, CD34+, CD90+ cells within the SVF of human breast tissue ( b , left panel). Representative images of unpurified cells in total SVF or FACS purified adipocyte precursors (APs) from mice ( a , right panel) or humans ( b , right panel) after culturing in adipogenic media for 8 days ( a ) or 13 days ( b ) and staining with Oil Red O. n = 3 biological repeats. c Schematic summarizing stages of adipogenesis. d Experimental strategy to measure EdU incorporation in adipocyte precursors by FACS (as in e and f ), and in mature adipocytes (as in g , h ). e Representative flow cytometry plots of EdU signal in APs (as defined in a ) in virgin mice and at involution days 1 and 3. f The average percentage of EdU+ APs identified with flow cytometry as in e . n = 4 mice per time point, P = 0.51 for virgin vs. involution day 1 data. g, h Representative image ( g ) and quantification ( h ) of EdU immunofluorescence in Adi p oq-Cre; mT/mG mouse injected with EdU according to the timeline in d . Note EdU+ (arrow) and EdU− nuclei (arrowhead) in mGFP+ adipocytes. White box indicates insets. n = 200–800 nuclei in each of four mice per group, P = 0.84. i Experimental strategy to inhibit adipogenesis during involution using GW9662, a pharmacological antagonist of PPARγ. j , k Representative image ( j ) and quantification ( k ) of MGs from GW9662- and vehicle-treated mice, stained with a perilipin antibody and DAPI. N = 8–17 fields in each of five mice per treatment group, P = 0.09. Scale bar is 50 μm in g and j . Data are mean ± SEM. Statistics were performed using a one-way ANOVA with Tukey’s multiple comparisons test ( f ) and a two-tailed unpaired t -test ( h , k ). **** P

    Article Snippet: Flow cytometry sorting and analysis For flow cytometry of primary murine cells, MG or skin tissue was minced and digested in Hanks Balanced Salt Solution (HBSS) (Sigma, H8264) supplemented with 3% BSA, 0.8 mg/ml collagenase type 2 (Worthington Biochemical, LS004174), 0.8 mM ZnCl2 , 1.0 mM MgCl2 and 1.2 mM CaCl2 for 75 min at 37 °C in a shaking water bath.

    Techniques: Flow Cytometry, Cytometry, FACS, Purification, Mouse Assay, Staining, Immunofluorescence, Injection, Two Tailed Test

    The pPAC7 vector . A) Schematic map of the pPAC7 vector which contains the EGFP gene cassette (EGFP), the blasticidin deaminase gene cassette (Bsr), the EBNA-1 gene cassette (EBNA-1), and the Epstein Barr virus origin of replication ( ori P). The pUC-link fragment is removed by BamH I or Not I digestion during the cloning process and replaced by large genomic DNA fragments allowing positive selection for recombinant clones on sucrose containing media due to the Sac BII gene present on the vector. The kanamycin resistance gene (Kan(R)) is a bacterial selection marker. The Not I restriction sites are used when verifying the different constructs. B) SK-BR-3 cells were transfected with pPAC7 and grown in the presence of blasticidin using the concentrations indicated. The percent of EGFP positive cells, from all viable cells, were measured by flow cytometry after 2, 4 and 6 days. The experiments were performed in triplicates with standard deviations shown. C) SK-BR-3 cells transfected with pPAC7 or pEGFP-C1 were sorted by flow cytometry and grown in non-selective medium. The percentage and total number of EGFP expressing cells was measured by flow cytometry 4, 11 and 15 days after cell sorting. The experiments were performed in triplicates with standard deviations indicated. D) The fluorescent microscopy pictures show the SK-BR-3 cells transfected with pEGFP-C1 and pPAC7 15 days after cell sorting. The shutter time is 1306 ms for pEGFP-C1 transfected and 390 ms for pPAC7 transfected cells.

    Journal: BMC Biotechnology

    Article Title: Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells

    doi: 10.1186/1472-6750-9-88

    Figure Lengend Snippet: The pPAC7 vector . A) Schematic map of the pPAC7 vector which contains the EGFP gene cassette (EGFP), the blasticidin deaminase gene cassette (Bsr), the EBNA-1 gene cassette (EBNA-1), and the Epstein Barr virus origin of replication ( ori P). The pUC-link fragment is removed by BamH I or Not I digestion during the cloning process and replaced by large genomic DNA fragments allowing positive selection for recombinant clones on sucrose containing media due to the Sac BII gene present on the vector. The kanamycin resistance gene (Kan(R)) is a bacterial selection marker. The Not I restriction sites are used when verifying the different constructs. B) SK-BR-3 cells were transfected with pPAC7 and grown in the presence of blasticidin using the concentrations indicated. The percent of EGFP positive cells, from all viable cells, were measured by flow cytometry after 2, 4 and 6 days. The experiments were performed in triplicates with standard deviations shown. C) SK-BR-3 cells transfected with pPAC7 or pEGFP-C1 were sorted by flow cytometry and grown in non-selective medium. The percentage and total number of EGFP expressing cells was measured by flow cytometry 4, 11 and 15 days after cell sorting. The experiments were performed in triplicates with standard deviations indicated. D) The fluorescent microscopy pictures show the SK-BR-3 cells transfected with pEGFP-C1 and pPAC7 15 days after cell sorting. The shutter time is 1306 ms for pEGFP-C1 transfected and 390 ms for pPAC7 transfected cells.

    Article Snippet: Flow cytometry Flow cytometry was performed using a Becton Dickinson dual-laser FACScalibur analysis instrument (BD Biosciences, CA, USA) and the Cell Quest software.

    Techniques: Plasmid Preparation, Clone Assay, Selection, Recombinant, Marker, Construct, Transfection, Flow Cytometry, Cytometry, Expressing, FACS, Microscopy, Mass Spectrometry

    Transfection efficiencies measured in 293T and SK-BR-3 cells . A) DNA from each of the constructs pPAC7, pPAC7-39 and pPAC7-CDH3 was digested with Not I and separated on a BioRad CHEF-mapper. During construction of pPAC7-39 one of the Not I restriction sites got deleted and thus giving a linearised fragment when digested with Not I. Selected fragments in the Low Range PFG Marker are indicated. B) Diagram showing the fraction of viable, EGFP-expressing 293T and SK-BR-3 cells measured by flow cytometry the day after the transfection with the following constructs: pPAC7, pPAC7-39 and pPAC7-CDH3. One μg of DNA from each construct was used for transfection, and the figure shows the transfection frequencies normalized with respect to the molar concentrations of plasmid DNA used in each transfection. For the 293T cell line, each bar represents three independent experiments all performed in triplicate. For the SK-BR-3 cell line each bar represents two separate experiments, both performed in triplicates. Standard deviations are indicated. C) Representative flow diagrams showing a 10-fold decrease in EGFP intensities in the 293T cell line with increasing plasmid size. Constructs used are: pPAC7, pPAC7-39 and pPAC7-CDH3.

    Journal: BMC Biotechnology

    Article Title: Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells

    doi: 10.1186/1472-6750-9-88

    Figure Lengend Snippet: Transfection efficiencies measured in 293T and SK-BR-3 cells . A) DNA from each of the constructs pPAC7, pPAC7-39 and pPAC7-CDH3 was digested with Not I and separated on a BioRad CHEF-mapper. During construction of pPAC7-39 one of the Not I restriction sites got deleted and thus giving a linearised fragment when digested with Not I. Selected fragments in the Low Range PFG Marker are indicated. B) Diagram showing the fraction of viable, EGFP-expressing 293T and SK-BR-3 cells measured by flow cytometry the day after the transfection with the following constructs: pPAC7, pPAC7-39 and pPAC7-CDH3. One μg of DNA from each construct was used for transfection, and the figure shows the transfection frequencies normalized with respect to the molar concentrations of plasmid DNA used in each transfection. For the 293T cell line, each bar represents three independent experiments all performed in triplicate. For the SK-BR-3 cell line each bar represents two separate experiments, both performed in triplicates. Standard deviations are indicated. C) Representative flow diagrams showing a 10-fold decrease in EGFP intensities in the 293T cell line with increasing plasmid size. Constructs used are: pPAC7, pPAC7-39 and pPAC7-CDH3.

    Article Snippet: Flow cytometry Flow cytometry was performed using a Becton Dickinson dual-laser FACScalibur analysis instrument (BD Biosciences, CA, USA) and the Cell Quest software.

    Techniques: Transfection, Construct, Marker, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation