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  • 99
    Thermo Fisher facs analysis
    Facs Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/facs analysis/product/Thermo Fisher
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    86
    Becton Dickinson facs analysis
    Induction of apoptosis by DHA/X-11 in AML cells with different levels of Bcl-xL and Mcl-1 NB4 and U937 cells were treated with DHA/X-11 at the indicated concentrations for 24 h. The apoptotic cells were determined by <t>PI/FACS</t> (A) and the protein levels were determined using Western blot analysis (B). HL60/M15 and HL60/V3 cells were treated with X-11 at 0.1 μM for 24 h and their extracts were used to measure the protein levels of Noxa and Mcl-1 with Western blotting (C) and the intact cells for percent of apoptosis by FACS after staining with <t>annexin</t> V (D).
    Facs Analysis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Beckman Coulter facs analysis
    The effect of phosphorylation or dephosphorylation on the CPT sensitivity of TopI activity in extracts from <t>FACS</t> separated CD44+ cells or CD44− <t>Caco2</t> cells. ( A ) The TopI activity in CD44+ cell extracts after phosphorylation or dephosphorylation. Whole cell extract was either left untreated (indicated by Control) or incubated with phosphatase (indicated by Dephos) or CK2 (indicated by Phos) before TopI activity was measured by REEAD in the presence of 60 µM CPT or 5% DMSO as indicated in the figure. One example randomly picked out of 12 individual microscopic images of each reaction sample is shown in the top panel. A quantitative depiction of three independent experiments, obtained as described in the legend of figure 1 is shown in the lower panel. ( B ) Same as ( A ), except that the analyses were performed on extracts from CD44− cells.
    Facs Analysis, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Becton Dickinson fluorescence activated cell sorter facs analysis
    Screening for tTA-expressing 293 cells. Human embryonic kidney 293 cell clones that stably expressed the tTA gene were transiently cotransfected with the pBIGF plasmid that produced the <t>GFP</t> under the control of the tetracycline-regulated promoter and the pcDNABFP plasmid that expressed the BFP under the control of the CMV promoter. Activation of the tetracycline-regulated promoter by the tTA in the presence and the absence of doxycycline (DOX) was evaluated by GFP expression and was determined by <t>FACS</t> analysis ( x axis). BFP expression from the CMV promoter served as a standard for transfection efficiency and transcription activity and was determined by FACS analysis ( y axis). Nontransfected human embryonic kidney 293 cells that expressed the tTA gene served as a negative control for either GFP or BFP expression.
    Fluorescence Activated Cell Sorter Facs Analysis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Induction of apoptosis by DHA/X-11 in AML cells with different levels of Bcl-xL and Mcl-1 NB4 and U937 cells were treated with DHA/X-11 at the indicated concentrations for 24 h. The apoptotic cells were determined by PI/FACS (A) and the protein levels were determined using Western blot analysis (B). HL60/M15 and HL60/V3 cells were treated with X-11 at 0.1 μM for 24 h and their extracts were used to measure the protein levels of Noxa and Mcl-1 with Western blotting (C) and the intact cells for percent of apoptosis by FACS after staining with annexin V (D).

    Journal: Oncotarget

    Article Title: Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety

    doi:

    Figure Lengend Snippet: Induction of apoptosis by DHA/X-11 in AML cells with different levels of Bcl-xL and Mcl-1 NB4 and U937 cells were treated with DHA/X-11 at the indicated concentrations for 24 h. The apoptotic cells were determined by PI/FACS (A) and the protein levels were determined using Western blot analysis (B). HL60/M15 and HL60/V3 cells were treated with X-11 at 0.1 μM for 24 h and their extracts were used to measure the protein levels of Noxa and Mcl-1 with Western blotting (C) and the intact cells for percent of apoptosis by FACS after staining with annexin V (D).

    Article Snippet: For FACS analysis with Annexin V staining, Annexin V–FITC Apoptosis Detection Kit and Annexin V–PI Apoptosis Detection Kit (BD Biosciences) were used to quantify apoptosis by FACS analysis.

    Techniques: FACS, Western Blot, Staining

    ABT-737 combined with X-11 synergistically induce apoptosis in U937 cells U937 cells were treated with ABT-737 (0.125 to 2 μM), X-11 (0.05 to 0.6 μM) or their combinations at different ratios for 24 h. Apoptotic cells were quantified based on morphologic changes by microscopic detection of AO/EB-stained cells (A). The nature of interaction between ABT-737 and X-11 was characterized by median dose effect analysis using CompuSyn software. CI values of less than 1.0 (horizontal line) correspond to a synergistic interaction (B). U937 cells were also treated with 0.5 μM ABT-737, 0.2 μM DHA and their combination for 24 h to determine the apoptosis induction by FACS after staining with annexin V-FITC (C) and protein regulation by Western blotting analysis (D).

    Journal: Oncotarget

    Article Title: Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety

    doi:

    Figure Lengend Snippet: ABT-737 combined with X-11 synergistically induce apoptosis in U937 cells U937 cells were treated with ABT-737 (0.125 to 2 μM), X-11 (0.05 to 0.6 μM) or their combinations at different ratios for 24 h. Apoptotic cells were quantified based on morphologic changes by microscopic detection of AO/EB-stained cells (A). The nature of interaction between ABT-737 and X-11 was characterized by median dose effect analysis using CompuSyn software. CI values of less than 1.0 (horizontal line) correspond to a synergistic interaction (B). U937 cells were also treated with 0.5 μM ABT-737, 0.2 μM DHA and their combination for 24 h to determine the apoptosis induction by FACS after staining with annexin V-FITC (C) and protein regulation by Western blotting analysis (D).

    Article Snippet: For FACS analysis with Annexin V staining, Annexin V–FITC Apoptosis Detection Kit and Annexin V–PI Apoptosis Detection Kit (BD Biosciences) were used to quantify apoptosis by FACS analysis.

    Techniques: Staining, Software, FACS, Western Blot

    ABT-737 combined with DHA synergistically induce apoptosis in U937 cells U937 cells were treated with ABT-737 (0.125 to 2 μM), DHA (0.2 to 2.4 μM) or their combinations at different ratios for 24 h. Apoptotic cells were quantified based on morphologic changes by microscopic detection of AO/EB-stained cells (A). The nature of interaction between ABT-737 and DHA was characterized by median dose effect analysis using CompuSyn software. CI values of less than 1.0 (horizontal line) correspond to a synergistic interaction. Fa on the x- axis denotes the fraction affected (B). U937 cells were treated with 0.5 μM ABT-737, 0.8 μM DHA and their combination for 24 h. The apoptotic cells were determined by FACS after staining with annexin V-FITC (C) and the relative levels of indicated proteins were determined by Western blotting using specific antibodies (D).

    Journal: Oncotarget

    Article Title: Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety

    doi:

    Figure Lengend Snippet: ABT-737 combined with DHA synergistically induce apoptosis in U937 cells U937 cells were treated with ABT-737 (0.125 to 2 μM), DHA (0.2 to 2.4 μM) or their combinations at different ratios for 24 h. Apoptotic cells were quantified based on morphologic changes by microscopic detection of AO/EB-stained cells (A). The nature of interaction between ABT-737 and DHA was characterized by median dose effect analysis using CompuSyn software. CI values of less than 1.0 (horizontal line) correspond to a synergistic interaction. Fa on the x- axis denotes the fraction affected (B). U937 cells were treated with 0.5 μM ABT-737, 0.8 μM DHA and their combination for 24 h. The apoptotic cells were determined by FACS after staining with annexin V-FITC (C) and the relative levels of indicated proteins were determined by Western blotting using specific antibodies (D).

    Article Snippet: For FACS analysis with Annexin V staining, Annexin V–FITC Apoptosis Detection Kit and Annexin V–PI Apoptosis Detection Kit (BD Biosciences) were used to quantify apoptosis by FACS analysis.

    Techniques: Staining, Software, FACS, Western Blot

    The combined effects of DHA/X-11 with ABT-737 in HL-60 cells which overexpress Bcl-2 HL-60/neo cells, (transfected with an empty vector), and HL-60/Bcl2, (transfected with a Bcl-2 expression vector), were treated with either DHA and X-11 alone or in combination with ABT-737 for 24 h. Percentages of apoptotic cells were determined using FACS after staining with PI (A) and the apoptosis related proteins were measured by Western blot analysis (B).

    Journal: Oncotarget

    Article Title: Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety

    doi:

    Figure Lengend Snippet: The combined effects of DHA/X-11 with ABT-737 in HL-60 cells which overexpress Bcl-2 HL-60/neo cells, (transfected with an empty vector), and HL-60/Bcl2, (transfected with a Bcl-2 expression vector), were treated with either DHA and X-11 alone or in combination with ABT-737 for 24 h. Percentages of apoptotic cells were determined using FACS after staining with PI (A) and the apoptosis related proteins were measured by Western blot analysis (B).

    Article Snippet: For FACS analysis with Annexin V staining, Annexin V–FITC Apoptosis Detection Kit and Annexin V–PI Apoptosis Detection Kit (BD Biosciences) were used to quantify apoptosis by FACS analysis.

    Techniques: Transfection, Plasmid Preparation, Expressing, FACS, Staining, Western Blot

    Superoxide (O) is induced by DHA and X-11 and contributes to apoptosis HL-60 cells pretreated with 1 μM DPI or not pre-treated, were exposed to 0.8 μM DHA or 0.2 μM X-11 for 15 h. The intracellular O 2 − content was determined by adding 5 μM MitoSOX TM Red followed by FACS analysis. The peak shift to the right indicates increase in the levels of O 2 − content (A). The effects of DPI on DHA and X-11-induced apoptosis were determined by PI staining followed by FACS analysis (B). Cleavage of PARP, caspase-3, -9, as well as Noxa and Bim levels were determined by Western blot using specific antibodies (C).

    Journal: Oncotarget

    Article Title: Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety

    doi:

    Figure Lengend Snippet: Superoxide (O) is induced by DHA and X-11 and contributes to apoptosis HL-60 cells pretreated with 1 μM DPI or not pre-treated, were exposed to 0.8 μM DHA or 0.2 μM X-11 for 15 h. The intracellular O 2 − content was determined by adding 5 μM MitoSOX TM Red followed by FACS analysis. The peak shift to the right indicates increase in the levels of O 2 − content (A). The effects of DPI on DHA and X-11-induced apoptosis were determined by PI staining followed by FACS analysis (B). Cleavage of PARP, caspase-3, -9, as well as Noxa and Bim levels were determined by Western blot using specific antibodies (C).

    Article Snippet: For FACS analysis with Annexin V staining, Annexin V–FITC Apoptosis Detection Kit and Annexin V–PI Apoptosis Detection Kit (BD Biosciences) were used to quantify apoptosis by FACS analysis.

    Techniques: FACS, Staining, Western Blot

    Iron is required for DHA and X-11-induce apoptosis and O 2 production HL-60 cells were pretreated for 4 h with 100 μM DFO, the iron chelator, or not pretreated, and then treated with DHA/X-11 for 24 h at the indicated concentrations. Cells were stained with PI and apoptotic cells determined by FACS analysis (A). Apoptosis related protein levels were measured by Western blot analyses (B). The levels of O 2 − were measured using MitoSOX TM Red by FACS (C).

    Journal: Oncotarget

    Article Title: Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety

    doi:

    Figure Lengend Snippet: Iron is required for DHA and X-11-induce apoptosis and O 2 production HL-60 cells were pretreated for 4 h with 100 μM DFO, the iron chelator, or not pretreated, and then treated with DHA/X-11 for 24 h at the indicated concentrations. Cells were stained with PI and apoptotic cells determined by FACS analysis (A). Apoptosis related protein levels were measured by Western blot analyses (B). The levels of O 2 − were measured using MitoSOX TM Red by FACS (C).

    Article Snippet: For FACS analysis with Annexin V staining, Annexin V–FITC Apoptosis Detection Kit and Annexin V–PI Apoptosis Detection Kit (BD Biosciences) were used to quantify apoptosis by FACS analysis.

    Techniques: Staining, FACS, Western Blot

    At low concentrations of DHA/X-11, an endoperoxide bridge is required for apoptosis induction (A), the chemical structures of DODHA and DOX-11 with the endoperoxide moiety containing only one oxygen. (B), the levels of O 2 − in HL-60 cells treated with DODHA/DOX-11. HL-60 cells untreated or treated with 0.8 μM DODHA or 0.2 μM DOX-11 for 15 h were used to determine the intracellular O 2 − content using MitoSOX TM Red by FACS. (C D), DODHA/DOX-11-induced apoptosis and protein changes. HL-60 cells were treated with DODHA, DOX-11, DHA or X-11 at the indicated concentrations for 24 h. The apoptotic cells were stained with PI and analyzed by FACS (C) and the levels of apoptosis related proteins were determined by Western blot analysis using specific antibodies (D).

    Journal: Oncotarget

    Article Title: Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety

    doi:

    Figure Lengend Snippet: At low concentrations of DHA/X-11, an endoperoxide bridge is required for apoptosis induction (A), the chemical structures of DODHA and DOX-11 with the endoperoxide moiety containing only one oxygen. (B), the levels of O 2 − in HL-60 cells treated with DODHA/DOX-11. HL-60 cells untreated or treated with 0.8 μM DODHA or 0.2 μM DOX-11 for 15 h were used to determine the intracellular O 2 − content using MitoSOX TM Red by FACS. (C D), DODHA/DOX-11-induced apoptosis and protein changes. HL-60 cells were treated with DODHA, DOX-11, DHA or X-11 at the indicated concentrations for 24 h. The apoptotic cells were stained with PI and analyzed by FACS (C) and the levels of apoptosis related proteins were determined by Western blot analysis using specific antibodies (D).

    Article Snippet: For FACS analysis with Annexin V staining, Annexin V–FITC Apoptosis Detection Kit and Annexin V–PI Apoptosis Detection Kit (BD Biosciences) were used to quantify apoptosis by FACS analysis.

    Techniques: FACS, Staining, Western Blot

    X-11 is more potent than DHA in apoptosis induction in HL-60 cells (A), chemical structures of DHA and X-11. (B), dose- and time-dependent apoptosis induction in HL-60 cells. Cells were treated with DHA and X-11 at the indicated concentrations for 12, 18 and 24 h. Percentages of apoptotic cells were determined based on morphological changes using a fluorescence microscope after staining with AO and EB. (C), percent of apoptotic HL-60 cells treated with DHA and X-11 for 24 h at the indicated concentrations. Apoptosis measured using staining with PI followed by FACS analysis and identification of the SubG1 population. AP, apoptotic cells; Con, control. (D), Western blot analyses of apoptosis-related proteins in HL-60 cells treated with DHA and X-11 for 24 h at the indicated concentrations. The relative levels of the proteins were determined by probing with specific antibodies. β-actin served as loading control.

    Journal: Oncotarget

    Article Title: Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety

    doi:

    Figure Lengend Snippet: X-11 is more potent than DHA in apoptosis induction in HL-60 cells (A), chemical structures of DHA and X-11. (B), dose- and time-dependent apoptosis induction in HL-60 cells. Cells were treated with DHA and X-11 at the indicated concentrations for 12, 18 and 24 h. Percentages of apoptotic cells were determined based on morphological changes using a fluorescence microscope after staining with AO and EB. (C), percent of apoptotic HL-60 cells treated with DHA and X-11 for 24 h at the indicated concentrations. Apoptosis measured using staining with PI followed by FACS analysis and identification of the SubG1 population. AP, apoptotic cells; Con, control. (D), Western blot analyses of apoptosis-related proteins in HL-60 cells treated with DHA and X-11 for 24 h at the indicated concentrations. The relative levels of the proteins were determined by probing with specific antibodies. β-actin served as loading control.

    Article Snippet: For FACS analysis with Annexin V staining, Annexin V–FITC Apoptosis Detection Kit and Annexin V–PI Apoptosis Detection Kit (BD Biosciences) were used to quantify apoptosis by FACS analysis.

    Techniques: Fluorescence, Microscopy, Staining, FACS, Western Blot

    The effect of phosphorylation or dephosphorylation on the CPT sensitivity of TopI activity in extracts from FACS separated CD44+ cells or CD44− Caco2 cells. ( A ) The TopI activity in CD44+ cell extracts after phosphorylation or dephosphorylation. Whole cell extract was either left untreated (indicated by Control) or incubated with phosphatase (indicated by Dephos) or CK2 (indicated by Phos) before TopI activity was measured by REEAD in the presence of 60 µM CPT or 5% DMSO as indicated in the figure. One example randomly picked out of 12 individual microscopic images of each reaction sample is shown in the top panel. A quantitative depiction of three independent experiments, obtained as described in the legend of figure 1 is shown in the lower panel. ( B ) Same as ( A ), except that the analyses were performed on extracts from CD44− cells.

    Journal: PLoS ONE

    Article Title: Decreased Camptothecin Sensitivity of the Stem-Cell-Like Fraction of Caco2 Cells Correlates with an Altered Phosphorylation Pattern of Topoisomerase I

    doi: 10.1371/journal.pone.0099628

    Figure Lengend Snippet: The effect of phosphorylation or dephosphorylation on the CPT sensitivity of TopI activity in extracts from FACS separated CD44+ cells or CD44− Caco2 cells. ( A ) The TopI activity in CD44+ cell extracts after phosphorylation or dephosphorylation. Whole cell extract was either left untreated (indicated by Control) or incubated with phosphatase (indicated by Dephos) or CK2 (indicated by Phos) before TopI activity was measured by REEAD in the presence of 60 µM CPT or 5% DMSO as indicated in the figure. One example randomly picked out of 12 individual microscopic images of each reaction sample is shown in the top panel. A quantitative depiction of three independent experiments, obtained as described in the legend of figure 1 is shown in the lower panel. ( B ) Same as ( A ), except that the analyses were performed on extracts from CD44− cells.

    Article Snippet: FACS analysis and cell sorting Caco2 cell subpopulations were counted or separated into two fractions by FACS using a Gallios Flow Cytometer (Beckman Coulter Danmark ApS) after staining with CD44-PE or CD133-PE antibodies.

    Techniques: De-Phosphorylation Assay, Cycling Probe Technology, Activity Assay, FACS, Incubation

    Phoshorylation pattern of TopI in extracts from CD44+ or CD44− cell subpopulations. ( A ) Two-dimensional silver staining (left panel) and immunoblot analysis (right panel) of TopI expression pattern in the Caco2 cell line. The positions of TopI are underlined. The TopI identity of two major isoforms recognized by TopI antibodies was ensured by the MS/MS analysis of several bands excised from the silver stained gel (data not shown). ( B ) One-dimentional immunoblot analysis of TopI expression in CD44− and CD44+ FACS sorted cell fractions by immunoblotting with TopI antibodies. β-actin was used as a loading control. ( C ) Two-dimentional immunoblot analysis of TopI expression and post-translational modification pattern in CD44− and CD44+ cell fractions (left-hand panels) by immunoblotting with TopI antibodies. Equal protein amounts were loaded on the gels. The time of blot exposure is identical. The resulting pattern of TopI after cell treatment with λ-PPase prior the 2D PAGE analysis is presented (right-hand panels). Arrowheads indicate multiple forms of TopI (black arrowheads – the position of the non-phosphorylated TopI, blue arrowheads – the positions of phosphorylated TopI isoforms; red arrowheads – the positions of non-phosphorylated otherwise modified isoforms). The positions of parental and modified TopI isoforms are verified by the superimposing of all analyzed blots.

    Journal: PLoS ONE

    Article Title: Decreased Camptothecin Sensitivity of the Stem-Cell-Like Fraction of Caco2 Cells Correlates with an Altered Phosphorylation Pattern of Topoisomerase I

    doi: 10.1371/journal.pone.0099628

    Figure Lengend Snippet: Phoshorylation pattern of TopI in extracts from CD44+ or CD44− cell subpopulations. ( A ) Two-dimensional silver staining (left panel) and immunoblot analysis (right panel) of TopI expression pattern in the Caco2 cell line. The positions of TopI are underlined. The TopI identity of two major isoforms recognized by TopI antibodies was ensured by the MS/MS analysis of several bands excised from the silver stained gel (data not shown). ( B ) One-dimentional immunoblot analysis of TopI expression in CD44− and CD44+ FACS sorted cell fractions by immunoblotting with TopI antibodies. β-actin was used as a loading control. ( C ) Two-dimentional immunoblot analysis of TopI expression and post-translational modification pattern in CD44− and CD44+ cell fractions (left-hand panels) by immunoblotting with TopI antibodies. Equal protein amounts were loaded on the gels. The time of blot exposure is identical. The resulting pattern of TopI after cell treatment with λ-PPase prior the 2D PAGE analysis is presented (right-hand panels). Arrowheads indicate multiple forms of TopI (black arrowheads – the position of the non-phosphorylated TopI, blue arrowheads – the positions of phosphorylated TopI isoforms; red arrowheads – the positions of non-phosphorylated otherwise modified isoforms). The positions of parental and modified TopI isoforms are verified by the superimposing of all analyzed blots.

    Article Snippet: FACS analysis and cell sorting Caco2 cell subpopulations were counted or separated into two fractions by FACS using a Gallios Flow Cytometer (Beckman Coulter Danmark ApS) after staining with CD44-PE or CD133-PE antibodies.

    Techniques: Silver Staining, Expressing, Mass Spectrometry, Staining, FACS, Modification, Polyacrylamide Gel Electrophoresis

    CPT response of CSC or non-CSC cell subpopulations of Caco2. ( A ) FACS analysis of Caco2 cells stained with PE-conjugated CD44 (top panel) or CD133 (lower panel) specific antibodies. ( B ) FACS analysis of Caco2 cells before or after FACS sorting based on CD44. (I) Unstained cells, (II) Caco2 cells stained for CD44 before sorting, (III) Caco2 cells after sorting for CD44+ cells, (IV) Caco2 cells after sorting for CD44− cells. The cells were analyzed after sorting by staining for CD44 ( C ) Survival assay of the FACS sorted CD44+ or CD44− cells. The cells were incubated with DMSO (the solvent of CPT) or 0.1, 0.2, or 0.4, µM of CPT for 60 hours before the percentage of viable cells was measured by the MTT method. ( D ) Graphical depiction of the REEAD assay. S(hTopI) folds into a dumbbell shaped structure, which supports cleavage and ligation activity mediated by TopI. Hereby the substrate is converted to a closed circle, which is hybridized to a surface-attached RCA-primer matching the specific primer annealing sequence (p) on the substrate. Subsequently, phi-polymerase is added to support RCA. The resulting RCA products are visualized by hybridization of fluorescently labeled probes matching the complementary identifier sequence (i) of the circles and the products analyzed using a fluorescence microscope. TopI is illustrated as a gray pacman with a “T”, the phi-polymerase as a dark gray circle. The RCA-primers and detection probes are indicated on the figure. ( E ) Measurement of TopI activity by REEAD in the CD44+ and CD44− FACS sorted cell populations, respectively. Representative microscopic images obtained by analyzing the TopI activity present in whole cell extracts from 10-10 4 cells are shown in the top panel. Signals obtained from circularized S(hTopI) are shown in red, while signals obtained from control circles are shown in green. The lower panel shows a quantitative depiction of three independent experiments. ( F ) CPT sensitivity of TopI in the sorted CD44+ and CD44− cells, respectively. S(hTopI) was incubated with whole cell extract from 10 4 cells in presence of 15 µM, 30 µM, 60 µM CPT or DMSO as indicated in the figure. One example out of 12 individual microscopic images of each reaction sample is shown in the top panel, where red signals correspond to circularized S(hTopI) while green signals were generated from control circles. The lower panel shows a quantitative depiction of three independent experiments. For all REEAD experiments, to avoid misinterpretation due to potential uneven distribution of RCA primers printed on the surface, the number of red and green signals were counted on 12 microscopic images and the results depicted as the ratio between the number of red (R) TopI specific- and green (G) control circle generated signals (R/G) as described [45] .

    Journal: PLoS ONE

    Article Title: Decreased Camptothecin Sensitivity of the Stem-Cell-Like Fraction of Caco2 Cells Correlates with an Altered Phosphorylation Pattern of Topoisomerase I

    doi: 10.1371/journal.pone.0099628

    Figure Lengend Snippet: CPT response of CSC or non-CSC cell subpopulations of Caco2. ( A ) FACS analysis of Caco2 cells stained with PE-conjugated CD44 (top panel) or CD133 (lower panel) specific antibodies. ( B ) FACS analysis of Caco2 cells before or after FACS sorting based on CD44. (I) Unstained cells, (II) Caco2 cells stained for CD44 before sorting, (III) Caco2 cells after sorting for CD44+ cells, (IV) Caco2 cells after sorting for CD44− cells. The cells were analyzed after sorting by staining for CD44 ( C ) Survival assay of the FACS sorted CD44+ or CD44− cells. The cells were incubated with DMSO (the solvent of CPT) or 0.1, 0.2, or 0.4, µM of CPT for 60 hours before the percentage of viable cells was measured by the MTT method. ( D ) Graphical depiction of the REEAD assay. S(hTopI) folds into a dumbbell shaped structure, which supports cleavage and ligation activity mediated by TopI. Hereby the substrate is converted to a closed circle, which is hybridized to a surface-attached RCA-primer matching the specific primer annealing sequence (p) on the substrate. Subsequently, phi-polymerase is added to support RCA. The resulting RCA products are visualized by hybridization of fluorescently labeled probes matching the complementary identifier sequence (i) of the circles and the products analyzed using a fluorescence microscope. TopI is illustrated as a gray pacman with a “T”, the phi-polymerase as a dark gray circle. The RCA-primers and detection probes are indicated on the figure. ( E ) Measurement of TopI activity by REEAD in the CD44+ and CD44− FACS sorted cell populations, respectively. Representative microscopic images obtained by analyzing the TopI activity present in whole cell extracts from 10-10 4 cells are shown in the top panel. Signals obtained from circularized S(hTopI) are shown in red, while signals obtained from control circles are shown in green. The lower panel shows a quantitative depiction of three independent experiments. ( F ) CPT sensitivity of TopI in the sorted CD44+ and CD44− cells, respectively. S(hTopI) was incubated with whole cell extract from 10 4 cells in presence of 15 µM, 30 µM, 60 µM CPT or DMSO as indicated in the figure. One example out of 12 individual microscopic images of each reaction sample is shown in the top panel, where red signals correspond to circularized S(hTopI) while green signals were generated from control circles. The lower panel shows a quantitative depiction of three independent experiments. For all REEAD experiments, to avoid misinterpretation due to potential uneven distribution of RCA primers printed on the surface, the number of red and green signals were counted on 12 microscopic images and the results depicted as the ratio between the number of red (R) TopI specific- and green (G) control circle generated signals (R/G) as described [45] .

    Article Snippet: FACS analysis and cell sorting Caco2 cell subpopulations were counted or separated into two fractions by FACS using a Gallios Flow Cytometer (Beckman Coulter Danmark ApS) after staining with CD44-PE or CD133-PE antibodies.

    Techniques: Cycling Probe Technology, FACS, Staining, Clonogenic Cell Survival Assay, Incubation, MTT Assay, Ligation, Activity Assay, Sequencing, Hybridization, Labeling, Fluorescence, Microscopy, Generated

    Screening for tTA-expressing 293 cells. Human embryonic kidney 293 cell clones that stably expressed the tTA gene were transiently cotransfected with the pBIGF plasmid that produced the GFP under the control of the tetracycline-regulated promoter and the pcDNABFP plasmid that expressed the BFP under the control of the CMV promoter. Activation of the tetracycline-regulated promoter by the tTA in the presence and the absence of doxycycline (DOX) was evaluated by GFP expression and was determined by FACS analysis ( x axis). BFP expression from the CMV promoter served as a standard for transfection efficiency and transcription activity and was determined by FACS analysis ( y axis). Nontransfected human embryonic kidney 293 cells that expressed the tTA gene served as a negative control for either GFP or BFP expression.

    Journal: Journal of Virology

    Article Title: A Packaging Cell Line for Lentivirus Vectors

    doi:

    Figure Lengend Snippet: Screening for tTA-expressing 293 cells. Human embryonic kidney 293 cell clones that stably expressed the tTA gene were transiently cotransfected with the pBIGF plasmid that produced the GFP under the control of the tetracycline-regulated promoter and the pcDNABFP plasmid that expressed the BFP under the control of the CMV promoter. Activation of the tetracycline-regulated promoter by the tTA in the presence and the absence of doxycycline (DOX) was evaluated by GFP expression and was determined by FACS analysis ( x axis). BFP expression from the CMV promoter served as a standard for transfection efficiency and transcription activity and was determined by FACS analysis ( y axis). Nontransfected human embryonic kidney 293 cells that expressed the tTA gene served as a negative control for either GFP or BFP expression.

    Article Snippet: Fluorescence-activated cell sorter (FACS) analysis for cellular GFP and BFP was performed by FACScan analysis (Becton Dickinson) with the CellQuest program (version 3.0.1f; Becton Dickinson).

    Techniques: Expressing, Clone Assay, Stable Transfection, Plasmid Preparation, Produced, Activation Assay, FACS, Transfection, Activity Assay, Negative Control

    Induction by sodium butyrate. SODk1Blue cells were induced by doxycycline (DOX) withdrawal in the presence or absence of sodium butyrate. Expression of the BFP and GFP was determined by FACS analysis ( y and x axis, respectively) and reflected the activation of the inducible HIV-1 packaging and the VSV-G envelope cassettes, respectively. Noninduced SODk1Blue cells served as a negative control.

    Journal: Journal of Virology

    Article Title: A Packaging Cell Line for Lentivirus Vectors

    doi:

    Figure Lengend Snippet: Induction by sodium butyrate. SODk1Blue cells were induced by doxycycline (DOX) withdrawal in the presence or absence of sodium butyrate. Expression of the BFP and GFP was determined by FACS analysis ( y and x axis, respectively) and reflected the activation of the inducible HIV-1 packaging and the VSV-G envelope cassettes, respectively. Noninduced SODk1Blue cells served as a negative control.

    Article Snippet: Fluorescence-activated cell sorter (FACS) analysis for cellular GFP and BFP was performed by FACScan analysis (Becton Dickinson) with the CellQuest program (version 3.0.1f; Becton Dickinson).

    Techniques: Expressing, FACS, Activation Assay, Negative Control

    Induction of SODk1Blue cells. SODk1Blue cells were induced by doxycycline withdrawal in the presence of sodium butyrate. Activation of the VSV-G envelope and the HIV-1 packaging cassettes was reflected by the expression levels of the GFP and the BFP genes, respectively, and was determined at days 0, 2, 3, and 4 postinduction by FACS analysis.

    Journal: Journal of Virology

    Article Title: A Packaging Cell Line for Lentivirus Vectors

    doi:

    Figure Lengend Snippet: Induction of SODk1Blue cells. SODk1Blue cells were induced by doxycycline withdrawal in the presence of sodium butyrate. Activation of the VSV-G envelope and the HIV-1 packaging cassettes was reflected by the expression levels of the GFP and the BFP genes, respectively, and was determined at days 0, 2, 3, and 4 postinduction by FACS analysis.

    Article Snippet: Fluorescence-activated cell sorter (FACS) analysis for cellular GFP and BFP was performed by FACScan analysis (Becton Dickinson) with the CellQuest program (version 3.0.1f; Becton Dickinson).

    Techniques: Activation Assay, Expressing, FACS