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    Millipore luciferase
    Phagocytosis activity in PLSCR1-depleted and -overexpressing cells. (A-C) THP-1 cells were transduced with lentiviruses expressing <t>shRNA</t> against PLSCR1 or Luciferase, treated with PMA for 72 h as indicated in Fig 4 , and then submitted to phagocytosis of IgG-opsonised SRBCs. (A) <t>shLuc-</t> (upper panels) or shPLSCR1-transduced (lower panels) cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa350-anti-rabbit IgG, anti-PLSCR1 (1E9 mAb) revealed with Alexa555-anti-mouse IgG, and Alexa488-phalloidin to reveal internalized SRBCs, PLSCR1 and F-actin, respectively. (B and C) shLuc- or shPLSCR1-transduced cells were incubated for 2, 5, 10 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-rabbit IgG. The number of external SRBCs and the total number of cell-associated SRBCs in each cell was counted for at least 100 cells in each experiment. In (B), results are expressed as the percentage of cell-associated (internal and external) SRBCs/cell relative to that measured in shLuc-transduced cells after 30 min of phagocytosis. In (C), results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis in shLuc-transduced control cells. (D and E) THP-1 cells were transfected with the vector for expression of HA-PLSCR1, and 18 hours later tested for phagocytosis of IgG-opsonised SRBCs. In (D), transfected cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa647-anti-rabbit IgG, anti-HA rat antibody revealed with Alexa488-anti-rat IgG, and Alexa555-phalloidin to reveal internalized SRBCs, HA-PLSCR1 and F-actin, respectively. In (E), cells were incubated for 5, 15 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-Rabbit. Cells were also stained with anti-HA rat antibody revealed with Alexa488-anti-rat IgG to identify transfected cells. The number of external SRBCs and the total number of cell-associated SRBCs in each cells were counted for at least 50 transfected cells and 50 non-transfected cells. Results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis for non-transfected control cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using students t test (n.s., p > 0.05; *, p
    Luciferase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 963 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phagocytosis activity in PLSCR1-depleted and -overexpressing cells. (A-C) THP-1 cells were transduced with lentiviruses expressing shRNA against PLSCR1 or Luciferase, treated with PMA for 72 h as indicated in Fig 4 , and then submitted to phagocytosis of IgG-opsonised SRBCs. (A) shLuc- (upper panels) or shPLSCR1-transduced (lower panels) cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa350-anti-rabbit IgG, anti-PLSCR1 (1E9 mAb) revealed with Alexa555-anti-mouse IgG, and Alexa488-phalloidin to reveal internalized SRBCs, PLSCR1 and F-actin, respectively. (B and C) shLuc- or shPLSCR1-transduced cells were incubated for 2, 5, 10 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-rabbit IgG. The number of external SRBCs and the total number of cell-associated SRBCs in each cell was counted for at least 100 cells in each experiment. In (B), results are expressed as the percentage of cell-associated (internal and external) SRBCs/cell relative to that measured in shLuc-transduced cells after 30 min of phagocytosis. In (C), results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis in shLuc-transduced control cells. (D and E) THP-1 cells were transfected with the vector for expression of HA-PLSCR1, and 18 hours later tested for phagocytosis of IgG-opsonised SRBCs. In (D), transfected cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa647-anti-rabbit IgG, anti-HA rat antibody revealed with Alexa488-anti-rat IgG, and Alexa555-phalloidin to reveal internalized SRBCs, HA-PLSCR1 and F-actin, respectively. In (E), cells were incubated for 5, 15 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-Rabbit. Cells were also stained with anti-HA rat antibody revealed with Alexa488-anti-rat IgG to identify transfected cells. The number of external SRBCs and the total number of cell-associated SRBCs in each cells were counted for at least 50 transfected cells and 50 non-transfected cells. Results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis for non-transfected control cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using students t test (n.s., p > 0.05; *, p

    Journal: PLoS ONE

    Article Title: Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages

    doi: 10.1371/journal.pone.0145617

    Figure Lengend Snippet: Phagocytosis activity in PLSCR1-depleted and -overexpressing cells. (A-C) THP-1 cells were transduced with lentiviruses expressing shRNA against PLSCR1 or Luciferase, treated with PMA for 72 h as indicated in Fig 4 , and then submitted to phagocytosis of IgG-opsonised SRBCs. (A) shLuc- (upper panels) or shPLSCR1-transduced (lower panels) cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa350-anti-rabbit IgG, anti-PLSCR1 (1E9 mAb) revealed with Alexa555-anti-mouse IgG, and Alexa488-phalloidin to reveal internalized SRBCs, PLSCR1 and F-actin, respectively. (B and C) shLuc- or shPLSCR1-transduced cells were incubated for 2, 5, 10 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-rabbit IgG. The number of external SRBCs and the total number of cell-associated SRBCs in each cell was counted for at least 100 cells in each experiment. In (B), results are expressed as the percentage of cell-associated (internal and external) SRBCs/cell relative to that measured in shLuc-transduced cells after 30 min of phagocytosis. In (C), results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis in shLuc-transduced control cells. (D and E) THP-1 cells were transfected with the vector for expression of HA-PLSCR1, and 18 hours later tested for phagocytosis of IgG-opsonised SRBCs. In (D), transfected cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa647-anti-rabbit IgG, anti-HA rat antibody revealed with Alexa488-anti-rat IgG, and Alexa555-phalloidin to reveal internalized SRBCs, HA-PLSCR1 and F-actin, respectively. In (E), cells were incubated for 5, 15 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-Rabbit. Cells were also stained with anti-HA rat antibody revealed with Alexa488-anti-rat IgG to identify transfected cells. The number of external SRBCs and the total number of cell-associated SRBCs in each cells were counted for at least 50 transfected cells and 50 non-transfected cells. Results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis for non-transfected control cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using students t test (n.s., p > 0.05; *, p

    Article Snippet: The pLKO.1 plasmid expressing shRNA against firefly luciferase (shLuc) (Sigma) was used to produce control particles.

    Techniques: Activity Assay, Transduction, Expressing, shRNA, Luciferase, Incubation, Staining, Labeling, Transfection, Plasmid Preparation

    Monocyte-to-macrophage differentiation and cell surface phosphatidylserine exposure in PLSCR1-depleted cells. THP-1 cells were transduced with lentiviruses expressing shRNA against either PLSCR1 or Luciferase used as a control, and then cultured for 72 h with or without PMA as previously. (A) Lysates from shRNA-transduced THP-1 cells were analyzed by Western blotting with anti-PLSCR1 (upper panels) and anti-ɣ-tubulin (lower panels). (B and C) Cell surface expression of CD14 and PS exposure. Treated or untreated shRNA-transduced THP-1 cells were stained with FITC-conjugated anti-CD14 antibodies (B) or PE-conjugated Annexin-V (C), and surface expression was measured by flow cytometry. Results are expressed as the percentage of the MFI relative to the non differentiated control shLuc-transduced cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using Student's t test (non significant, p > 0.05).

    Journal: PLoS ONE

    Article Title: Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages

    doi: 10.1371/journal.pone.0145617

    Figure Lengend Snippet: Monocyte-to-macrophage differentiation and cell surface phosphatidylserine exposure in PLSCR1-depleted cells. THP-1 cells were transduced with lentiviruses expressing shRNA against either PLSCR1 or Luciferase used as a control, and then cultured for 72 h with or without PMA as previously. (A) Lysates from shRNA-transduced THP-1 cells were analyzed by Western blotting with anti-PLSCR1 (upper panels) and anti-ɣ-tubulin (lower panels). (B and C) Cell surface expression of CD14 and PS exposure. Treated or untreated shRNA-transduced THP-1 cells were stained with FITC-conjugated anti-CD14 antibodies (B) or PE-conjugated Annexin-V (C), and surface expression was measured by flow cytometry. Results are expressed as the percentage of the MFI relative to the non differentiated control shLuc-transduced cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using Student's t test (non significant, p > 0.05).

    Article Snippet: The pLKO.1 plasmid expressing shRNA against firefly luciferase (shLuc) (Sigma) was used to produce control particles.

    Techniques: Transduction, Expressing, shRNA, Luciferase, Cell Culture, Western Blot, Staining, Flow Cytometry, Cytometry

    Regulation of the Wnt/β-catenin pathway by the α6A variant subunit. Representative WB and graph of the densiometric analysis for the detection of active β-catenin and total β-catenin in the whole cell extract ( A ) and β-catenin in nuclear extracts ( B ). β-Actin served as loading control in cell extracts and histone H1 for nuclear extracts. Statistical analysis between shctrl and shα6A: * P ≤ 0.05, ** P ≤ 0.01, t -test, n = 3. ( C ) TOPflash assay of the response of β-catenin/TCF4 promotor activity in the α6A variant knocked down cell lines and their corresponding shctrl. Results showed the net luciferase/renilla ratio (Topflash − FOPflash). Statistical analysis between shctrl and shα6A: * P ≤ 0.05, ** P ≤ 0.01, t -test, n = 3.

    Journal: Carcinogenesis

    Article Title: Integrin ?6A splice variant regulates proliferation and the Wnt/?-catenin pathway in human colorectal cancer cells

    doi: 10.1093/carcin/bgu006

    Figure Lengend Snippet: Regulation of the Wnt/β-catenin pathway by the α6A variant subunit. Representative WB and graph of the densiometric analysis for the detection of active β-catenin and total β-catenin in the whole cell extract ( A ) and β-catenin in nuclear extracts ( B ). β-Actin served as loading control in cell extracts and histone H1 for nuclear extracts. Statistical analysis between shctrl and shα6A: * P ≤ 0.05, ** P ≤ 0.01, t -test, n = 3. ( C ) TOPflash assay of the response of β-catenin/TCF4 promotor activity in the α6A variant knocked down cell lines and their corresponding shctrl. Results showed the net luciferase/renilla ratio (Topflash − FOPflash). Statistical analysis between shctrl and shα6A: * P ≤ 0.05, ** P ≤ 0.01, t -test, n = 3.

    Article Snippet: Transfections and luciferase assays TOPflash and FOPflash reporter plasmids (Millipore) were transfected into CRC cell lines with Effectene transfection reagent (Qiagen) using the manufacturer’s instructions.

    Techniques: Variant Assay, Western Blot, TOPFlash assay, Activity Assay, Luciferase