countbright solution  (Thermo Fisher)


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    Structured Review

    Thermo Fisher countbright solution
    Dendritic cell sensing of NGA formulations . ( a ) CD172a + sorted cells (0.5 × 10 6 cells/well; prelabeled with anti-CD172a and anti-CD14 antibodies) were placed in a 3 µm filter transwell inserts, and added into wells of 24-well companion plates preseeded with the NGA formulations (10% v/v in phenol red-free DMEM) or controls as shown on the x-axis. Following incubation at 39 °C for 3 hours, the cells which had migrated through the filters into the chamber containing the NGA formulations were assessed by flow cytometry. Quantification of the migrated cells was performed by counting a reference of 5000 <t>CountBright</t> beads to obtain the number of migrating cells/cm 3 . This was related to the number of migrating cells obtained with the medium control to give the migration index. ( b ) CD172a + sorted cells were incubated either alone (“negative”), with NGA (“NGA alone”), with NGA carrying unlabeled or labeled RepRNA (“NGA-RepRNA unlabeled”; “NGA-RepRNA FITC-labeled”), or with NGA-Lipo carrying labeled RepRNA (“NGA-Lipo RepRNA”). Following incubation in DMEM/1% (v/v) porcine serum for 2 or 16 hours at 39 °C, the forward and side scatter profiles (FSC, SSC) of the cells was determined by flow cytometry. The results show an example for the 16 hours incubation time point from 10 separate experiments; similar results were obtained with the 2 hours incubation time point.
    Countbright Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1764 article reviews
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    Images

    1) Product Images from "Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo"

    Article Title: Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2014.24

    Dendritic cell sensing of NGA formulations . ( a ) CD172a + sorted cells (0.5 × 10 6 cells/well; prelabeled with anti-CD172a and anti-CD14 antibodies) were placed in a 3 µm filter transwell inserts, and added into wells of 24-well companion plates preseeded with the NGA formulations (10% v/v in phenol red-free DMEM) or controls as shown on the x-axis. Following incubation at 39 °C for 3 hours, the cells which had migrated through the filters into the chamber containing the NGA formulations were assessed by flow cytometry. Quantification of the migrated cells was performed by counting a reference of 5000 CountBright beads to obtain the number of migrating cells/cm 3 . This was related to the number of migrating cells obtained with the medium control to give the migration index. ( b ) CD172a + sorted cells were incubated either alone (“negative”), with NGA (“NGA alone”), with NGA carrying unlabeled or labeled RepRNA (“NGA-RepRNA unlabeled”; “NGA-RepRNA FITC-labeled”), or with NGA-Lipo carrying labeled RepRNA (“NGA-Lipo RepRNA”). Following incubation in DMEM/1% (v/v) porcine serum for 2 or 16 hours at 39 °C, the forward and side scatter profiles (FSC, SSC) of the cells was determined by flow cytometry. The results show an example for the 16 hours incubation time point from 10 separate experiments; similar results were obtained with the 2 hours incubation time point.
    Figure Legend Snippet: Dendritic cell sensing of NGA formulations . ( a ) CD172a + sorted cells (0.5 × 10 6 cells/well; prelabeled with anti-CD172a and anti-CD14 antibodies) were placed in a 3 µm filter transwell inserts, and added into wells of 24-well companion plates preseeded with the NGA formulations (10% v/v in phenol red-free DMEM) or controls as shown on the x-axis. Following incubation at 39 °C for 3 hours, the cells which had migrated through the filters into the chamber containing the NGA formulations were assessed by flow cytometry. Quantification of the migrated cells was performed by counting a reference of 5000 CountBright beads to obtain the number of migrating cells/cm 3 . This was related to the number of migrating cells obtained with the medium control to give the migration index. ( b ) CD172a + sorted cells were incubated either alone (“negative”), with NGA (“NGA alone”), with NGA carrying unlabeled or labeled RepRNA (“NGA-RepRNA unlabeled”; “NGA-RepRNA FITC-labeled”), or with NGA-Lipo carrying labeled RepRNA (“NGA-Lipo RepRNA”). Following incubation in DMEM/1% (v/v) porcine serum for 2 or 16 hours at 39 °C, the forward and side scatter profiles (FSC, SSC) of the cells was determined by flow cytometry. The results show an example for the 16 hours incubation time point from 10 separate experiments; similar results were obtained with the 2 hours incubation time point.

    Techniques Used: Incubation, Flow Cytometry, Cytometry, Migration, Labeling

    2) Product Images from "Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells"

    Article Title: Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells

    Journal: Scientific Reports

    doi: 10.1038/srep32034

    Osteoblast-EVs stimulate the proliferation of the immature cells. (a,b) Sorted phenotypic HSCs were loaded with CellTrace TM Violet and incubated in the absence (control) or presence of osteoblast-EVs for 5 days. Flow cytometry plots show the distribution of the progeny of ( a ) CD34 + progenitors and ( b ) CD90 + phenotypic HSCs. (c,d) The percentages (mean ± SD) of cells that have divided 1–4 times within ( c ) CD34 + progenitors and ( d ) CD90 + phenotypic HSCs (N = 2).
    Figure Legend Snippet: Osteoblast-EVs stimulate the proliferation of the immature cells. (a,b) Sorted phenotypic HSCs were loaded with CellTrace TM Violet and incubated in the absence (control) or presence of osteoblast-EVs for 5 days. Flow cytometry plots show the distribution of the progeny of ( a ) CD34 + progenitors and ( b ) CD90 + phenotypic HSCs. (c,d) The percentages (mean ± SD) of cells that have divided 1–4 times within ( c ) CD34 + progenitors and ( d ) CD90 + phenotypic HSCs (N = 2).

    Techniques Used: Incubation, Flow Cytometry, Cytometry

    3) Product Images from "Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells"

    Article Title: Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells

    Journal: Scientific Reports

    doi: 10.1038/srep32034

    Osteoblast-EVs stimulate the proliferation of the immature cells. (a,b) Sorted phenotypic HSCs were loaded with CellTrace TM Violet and incubated in the absence (control) or presence of osteoblast-EVs for 5 days. Flow cytometry plots show the distribution of the progeny of ( a ) CD34 + progenitors and ( b ) CD90 + phenotypic HSCs. (c,d) The percentages (mean ± SD) of cells that have divided 1–4 times within ( c ) CD34 + progenitors and ( d ) CD90 + phenotypic HSCs (N = 2).
    Figure Legend Snippet: Osteoblast-EVs stimulate the proliferation of the immature cells. (a,b) Sorted phenotypic HSCs were loaded with CellTrace TM Violet and incubated in the absence (control) or presence of osteoblast-EVs for 5 days. Flow cytometry plots show the distribution of the progeny of ( a ) CD34 + progenitors and ( b ) CD90 + phenotypic HSCs. (c,d) The percentages (mean ± SD) of cells that have divided 1–4 times within ( c ) CD34 + progenitors and ( d ) CD90 + phenotypic HSCs (N = 2).

    Techniques Used: Incubation, Flow Cytometry, Cytometry

    Related Articles

    Flow Cytometry:

    Article Title: Bone marrow stromal cells from multiple myeloma patients uniquely induce bortezomib resistant NF-?B activity in myeloma cells
    Article Snippet: .. Flow Cytometry Antibodies used were anti-CD31 Allophycocyanin (APC), anti-CD45 Phycoerythrin (PE), anti-CD90 APC (eBioscience, San Diego, CA, USA), anti-CD 54 Fluorescein isothiocyanate (FITC), anti-CD105 APC, anti-HLA-ABC FITC (Invitrogen, Carlsbad, CA, USA), anti-CD14 FITC, anti-CD29 PE, anti-CD34 APC, anti-CD44 PE, anti-CD73 PE, anti-HLA-ABC FITC, anti-HLA-DR FITC (BD Pharmingen, San Diego, CA, USA), IgG2a FITC isotype control antibody, IgG1 PE isotype control antibody, IgG1 APC isotype control antibody (Miltenyi Biotech). .. For surface staining, cells were harvested by trypsinization for two minutes at 37°C.

    Article Title: Spheroid Culture of Mesenchymal Stromal Cells Results in Morphorheological Properties Appropriate for Improved Microcirculation
    Article Snippet: .. Flow Cytometry : The following antibodies were used: anti‐CD45‐PECy7 (BD, Franklin Lakes, NJ), anti‐CD90‐APC (eBioscience Inc., San Diego, CA), anti‐CD105‐APC, anti‐CD34‐PE, anti‐HLA‐DR‐APC, anti‐CD73‐PE, anti‐CD14‐APC, anti‐CD19‐APC, and anti‐CD133‐FITC (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). .. To evaluate proliferation and apoptosis, carboxyfluorescein diacetate succinimidyl ester (CFSE) staining (Thermofisher Scientific, Waltham, MA) and propidium iodide (PI) staining (eBioscience Inc., San Diego, CA) were performed, respectively, according to the protocols of the manufacturers.

    Fluorescence:

    Article Title: Allogenic Adipose Tissue-Derived Mesenchymal Stem Cells Ameliorate Acute Hepatic Injury in Dogs
    Article Snippet: .. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 105 cells/tube), washed with FACS buffer (PBS containing 2% FBS), blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated with the following fluorescein- (FITC-) or phycoerythrin- (PE-) conjugated antibodies: anti-CD14-FITC (BD PharMingen), anti-CD29-PE (BioLegend), anti-CD34-PE (R & D Systems), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience), and anti-CD90-PE (eBioscience) or their respective isotype controls listed in . .. The cells were washed twice with FACS buffer and resuspended in 500 μ l FACS buffer.

    Article Title: Comparison of Properties of Stem Cells Isolated from Adipose Tissue and Lipomas in Dogs
    Article Snippet: .. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 105 cells/tube), washed with FACS buffer (PBS containing 2% FBS), and then incubated with the following fluorescein (FITC)- or phycoerythrin (PE)-conjugated antibodies: anti-CD14-FITC (clone M5E2; BD Pharmingen), anti-CD29-PE (clone TS2/16; BioLegend), anti-CD34-PE (clone 1H6; R & D Systems), anti-CD44-PE (clone IM7; BioLegend), anti-CD45-FITC (clone YKIX716.13; eBioscience), and anti-CD90-PE (clone YKIX337.217; eBioscience) or their respective isotype controls [ , ]. .. The cells were washed twice with FACS buffer and resuspended in 500 μ l FACS buffer.

    Cytometry:

    Article Title: Bone marrow stromal cells from multiple myeloma patients uniquely induce bortezomib resistant NF-?B activity in myeloma cells
    Article Snippet: .. Flow Cytometry Antibodies used were anti-CD31 Allophycocyanin (APC), anti-CD45 Phycoerythrin (PE), anti-CD90 APC (eBioscience, San Diego, CA, USA), anti-CD 54 Fluorescein isothiocyanate (FITC), anti-CD105 APC, anti-HLA-ABC FITC (Invitrogen, Carlsbad, CA, USA), anti-CD14 FITC, anti-CD29 PE, anti-CD34 APC, anti-CD44 PE, anti-CD73 PE, anti-HLA-ABC FITC, anti-HLA-DR FITC (BD Pharmingen, San Diego, CA, USA), IgG2a FITC isotype control antibody, IgG1 PE isotype control antibody, IgG1 APC isotype control antibody (Miltenyi Biotech). .. For surface staining, cells were harvested by trypsinization for two minutes at 37°C.

    Article Title: Spheroid Culture of Mesenchymal Stromal Cells Results in Morphorheological Properties Appropriate for Improved Microcirculation
    Article Snippet: .. Flow Cytometry : The following antibodies were used: anti‐CD45‐PECy7 (BD, Franklin Lakes, NJ), anti‐CD90‐APC (eBioscience Inc., San Diego, CA), anti‐CD105‐APC, anti‐CD34‐PE, anti‐HLA‐DR‐APC, anti‐CD73‐PE, anti‐CD14‐APC, anti‐CD19‐APC, and anti‐CD133‐FITC (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). .. To evaluate proliferation and apoptosis, carboxyfluorescein diacetate succinimidyl ester (CFSE) staining (Thermofisher Scientific, Waltham, MA) and propidium iodide (PI) staining (eBioscience Inc., San Diego, CA) were performed, respectively, according to the protocols of the manufacturers.

    Blocking Assay:

    Article Title: Allogenic Adipose Tissue-Derived Mesenchymal Stem Cells Ameliorate Acute Hepatic Injury in Dogs
    Article Snippet: .. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 105 cells/tube), washed with FACS buffer (PBS containing 2% FBS), blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated with the following fluorescein- (FITC-) or phycoerythrin- (PE-) conjugated antibodies: anti-CD14-FITC (BD PharMingen), anti-CD29-PE (BioLegend), anti-CD34-PE (R & D Systems), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience), and anti-CD90-PE (eBioscience) or their respective isotype controls listed in . .. The cells were washed twice with FACS buffer and resuspended in 500 μ l FACS buffer.

    Incubation:

    Article Title: Tissue Factor Signals Airway Epithelial Basal Cell Survival via Coagulation and Protease-Activated Receptor Isoforms 1 and 2
    Article Snippet: .. One million passage 1 cells were incubated with anti–TF-FITC (American Diagnostica, Stamford, CT), anti-CD31-APC (eBioscience, San Diego CA) (to exclude endothelial cells), anti–CD45-APC (eBioscience) (to exclude hematopoietic cells), and anti–CD90-APC (eBioscience) (to exclude fibroblasts) antibodies diluted in PBS containing BSA (1%) for 30 minutes at 4°C. ..

    Article Title: Allogenic Adipose Tissue-Derived Mesenchymal Stem Cells Ameliorate Acute Hepatic Injury in Dogs
    Article Snippet: .. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 105 cells/tube), washed with FACS buffer (PBS containing 2% FBS), blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated with the following fluorescein- (FITC-) or phycoerythrin- (PE-) conjugated antibodies: anti-CD14-FITC (BD PharMingen), anti-CD29-PE (BioLegend), anti-CD34-PE (R & D Systems), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience), and anti-CD90-PE (eBioscience) or their respective isotype controls listed in . .. The cells were washed twice with FACS buffer and resuspended in 500 μ l FACS buffer.

    Article Title: Comparison of Properties of Stem Cells Isolated from Adipose Tissue and Lipomas in Dogs
    Article Snippet: .. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 105 cells/tube), washed with FACS buffer (PBS containing 2% FBS), and then incubated with the following fluorescein (FITC)- or phycoerythrin (PE)-conjugated antibodies: anti-CD14-FITC (clone M5E2; BD Pharmingen), anti-CD29-PE (clone TS2/16; BioLegend), anti-CD34-PE (clone 1H6; R & D Systems), anti-CD44-PE (clone IM7; BioLegend), anti-CD45-FITC (clone YKIX716.13; eBioscience), and anti-CD90-PE (clone YKIX337.217; eBioscience) or their respective isotype controls [ , ]. .. The cells were washed twice with FACS buffer and resuspended in 500 μ l FACS buffer.

    Staining:

    Article Title: Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures
    Article Snippet: .. MACS-selected CD34+ cells were either used directly in experiments or stained with anti-Lin-FITC, anti-CD38-PerCP-Cy5.5, anti-CD90-PE (all from eBioscience, Vienna, Austria), anti-CD34-PE-Cy7, anti-CD45RA-APC-H7 (both from BD Biosciences, San Jose, CA, USA) and DAPI (Sigma-Aldrich, St Louis, MO, USA) after which viable DAPI- Lin- CD34+ CD38low CD45RAlow CD90+ -cells, highly enriched for hematopoietic stem cells (HSC)[ ], were sorted using BD FACSAria Cell Sorting System (BD Biosciences, San Jose, CA, USA). .. Expansion cultures Selected CD34+ cells and sorted DAPI- Lin- CD34+ CD38low CD45RAlow CD90+ -cellswere cultured in serum free Glycostem Basic Growth Medium (GBGM, Glycostem, Oss, The Netherlands) or StemSpan Serum-Free Expansion Medium (SFEM, Stemcell Technologies, Grenoble, France) supplemented with 20 μg/ml low molecular weight heparin (Abbott, Wiesbaden, Germany) and the early acting growth factors SCF (50 ng/ml, Cellgenix, Freiburg, Germany), Flt3L (50 ng/ml, Cellgenix, Freiburg, Germany) and TPO (50 ng/ml, Cellgenix, Freiburg, Germany) (from now on referred to as ‘SFT medium’) with or without the addition of 250 ng/ml purified Wnt3a unless indicated otherwise.

    FACS:

    Article Title: Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures
    Article Snippet: .. MACS-selected CD34+ cells were either used directly in experiments or stained with anti-Lin-FITC, anti-CD38-PerCP-Cy5.5, anti-CD90-PE (all from eBioscience, Vienna, Austria), anti-CD34-PE-Cy7, anti-CD45RA-APC-H7 (both from BD Biosciences, San Jose, CA, USA) and DAPI (Sigma-Aldrich, St Louis, MO, USA) after which viable DAPI- Lin- CD34+ CD38low CD45RAlow CD90+ -cells, highly enriched for hematopoietic stem cells (HSC)[ ], were sorted using BD FACSAria Cell Sorting System (BD Biosciences, San Jose, CA, USA). .. Expansion cultures Selected CD34+ cells and sorted DAPI- Lin- CD34+ CD38low CD45RAlow CD90+ -cellswere cultured in serum free Glycostem Basic Growth Medium (GBGM, Glycostem, Oss, The Netherlands) or StemSpan Serum-Free Expansion Medium (SFEM, Stemcell Technologies, Grenoble, France) supplemented with 20 μg/ml low molecular weight heparin (Abbott, Wiesbaden, Germany) and the early acting growth factors SCF (50 ng/ml, Cellgenix, Freiburg, Germany), Flt3L (50 ng/ml, Cellgenix, Freiburg, Germany) and TPO (50 ng/ml, Cellgenix, Freiburg, Germany) (from now on referred to as ‘SFT medium’) with or without the addition of 250 ng/ml purified Wnt3a unless indicated otherwise.

    Article Title: Comparison of Properties of Stem Cells Isolated from Adipose Tissue and Lipomas in Dogs
    Article Snippet: .. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 105 cells/tube), washed with FACS buffer (PBS containing 2% FBS), and then incubated with the following fluorescein (FITC)- or phycoerythrin (PE)-conjugated antibodies: anti-CD14-FITC (clone M5E2; BD Pharmingen), anti-CD29-PE (clone TS2/16; BioLegend), anti-CD34-PE (clone 1H6; R & D Systems), anti-CD44-PE (clone IM7; BioLegend), anti-CD45-FITC (clone YKIX716.13; eBioscience), and anti-CD90-PE (clone YKIX337.217; eBioscience) or their respective isotype controls [ , ]. .. The cells were washed twice with FACS buffer and resuspended in 500 μ l FACS buffer.

    Binding Assay:

    Article Title: Allogenic Adipose Tissue-Derived Mesenchymal Stem Cells Ameliorate Acute Hepatic Injury in Dogs
    Article Snippet: .. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 105 cells/tube), washed with FACS buffer (PBS containing 2% FBS), blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated with the following fluorescein- (FITC-) or phycoerythrin- (PE-) conjugated antibodies: anti-CD14-FITC (BD PharMingen), anti-CD29-PE (BioLegend), anti-CD34-PE (R & D Systems), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience), and anti-CD90-PE (eBioscience) or their respective isotype controls listed in . .. The cells were washed twice with FACS buffer and resuspended in 500 μ l FACS buffer.

    Magnetic Cell Separation:

    Article Title: Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures
    Article Snippet: .. MACS-selected CD34+ cells were either used directly in experiments or stained with anti-Lin-FITC, anti-CD38-PerCP-Cy5.5, anti-CD90-PE (all from eBioscience, Vienna, Austria), anti-CD34-PE-Cy7, anti-CD45RA-APC-H7 (both from BD Biosciences, San Jose, CA, USA) and DAPI (Sigma-Aldrich, St Louis, MO, USA) after which viable DAPI- Lin- CD34+ CD38low CD45RAlow CD90+ -cells, highly enriched for hematopoietic stem cells (HSC)[ ], were sorted using BD FACSAria Cell Sorting System (BD Biosciences, San Jose, CA, USA). .. Expansion cultures Selected CD34+ cells and sorted DAPI- Lin- CD34+ CD38low CD45RAlow CD90+ -cellswere cultured in serum free Glycostem Basic Growth Medium (GBGM, Glycostem, Oss, The Netherlands) or StemSpan Serum-Free Expansion Medium (SFEM, Stemcell Technologies, Grenoble, France) supplemented with 20 μg/ml low molecular weight heparin (Abbott, Wiesbaden, Germany) and the early acting growth factors SCF (50 ng/ml, Cellgenix, Freiburg, Germany), Flt3L (50 ng/ml, Cellgenix, Freiburg, Germany) and TPO (50 ng/ml, Cellgenix, Freiburg, Germany) (from now on referred to as ‘SFT medium’) with or without the addition of 250 ng/ml purified Wnt3a unless indicated otherwise.

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  • 91
    Thermo Fisher hiv 1 capsid protein
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    Altered phosphorylation signatures observed in a latently infected Jurkat cell line model following T cell activation. ( a ) Schematic depicts the sorting strategy for generating a clonal DHIV-GFP/ HIV-1 LAI (X4)-infected Jurkat cell line (J-DHIV). ( b ) Histogram of GFP expression in the J-DHIV cells before (black) and after stimulation with PMA/I (yellow) measured by flow cytometry. ( c ) Viral reactivation levels after 24 hours of stimulation across a panel of LRAs. Data presented as means ± SD for 3 biological replicates. ( d , e ) Heatmaps depict phospho-protein time courses following CD3/CD28 or PMA/I stimulation in Jurkat and J-DHIV cells over 24 hours measured by bead-based immunoassay. Phospho-signals were measured in biological triplicate and 90% of measurements had an SD

    Journal: Scientific Reports

    Article Title: Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells

    doi: 10.1038/s41598-017-15532-0

    Figure Lengend Snippet: Altered phosphorylation signatures observed in a latently infected Jurkat cell line model following T cell activation. ( a ) Schematic depicts the sorting strategy for generating a clonal DHIV-GFP/ HIV-1 LAI (X4)-infected Jurkat cell line (J-DHIV). ( b ) Histogram of GFP expression in the J-DHIV cells before (black) and after stimulation with PMA/I (yellow) measured by flow cytometry. ( c ) Viral reactivation levels after 24 hours of stimulation across a panel of LRAs. Data presented as means ± SD for 3 biological replicates. ( d , e ) Heatmaps depict phospho-protein time courses following CD3/CD28 or PMA/I stimulation in Jurkat and J-DHIV cells over 24 hours measured by bead-based immunoassay. Phospho-signals were measured in biological triplicate and 90% of measurements had an SD

    Article Snippet: For primary cells, viral activation was measured by intracellular staining for p24, an HIV-1 capsid protein, with the monoclonal antibody AG3.0 at a 1:40 dilution (NIH AIDS Reagent Program, courtesy of Dr. Jonathan Allan) and anti-mouse IgG secondary antibodies conjugated to AlexaFluor 647 (ThermoFisher Scientific).

    Techniques: Infection, Activation Assay, Expressing, Flow Cytometry, Cytometry, Bead-based Assay

    Latently infected CD4+ T CM cells exhibit altered phosphorylation signatures relative to uninfected cells following T cell activation. ( a ) Experimental timeline of in vitro primary CD4+ T CM cell model generation. After DHIV-Nef +/ HIV-1 LAI (X4) infection on day 5, uninfected and infected cells are cultured in parallel. ( b ) Scatter plots of Gag p24 expression in CD4+ T CM cells before (left) and after stimulation with CD3/CD28 (right) measured by flow cytometry. Representative data from donor 2; n = 10,000 cells. ( c ) Range of latent infection levels for replicate infections across 3 donors as estimated by CD3/CD28 stimulation of viral reactivation. ( d ) Viral reactivation after 48 hours of stimulation across a panel of LRAs. Data presented as means ± SD (n = 3) for donor 2. ( e , f ) Heatmaps depict phospho-protein time courses following CD3/CD28 ( e ) or PMA/I stimulation ( f ) in uninfected and infected CD4+ T CM cells for 3 donors over 48 hours measured by bead-based immunoassay. Phospho-signals were measured in biological triplicate and 90% of measurements had an SD

    Journal: Scientific Reports

    Article Title: Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells

    doi: 10.1038/s41598-017-15532-0

    Figure Lengend Snippet: Latently infected CD4+ T CM cells exhibit altered phosphorylation signatures relative to uninfected cells following T cell activation. ( a ) Experimental timeline of in vitro primary CD4+ T CM cell model generation. After DHIV-Nef +/ HIV-1 LAI (X4) infection on day 5, uninfected and infected cells are cultured in parallel. ( b ) Scatter plots of Gag p24 expression in CD4+ T CM cells before (left) and after stimulation with CD3/CD28 (right) measured by flow cytometry. Representative data from donor 2; n = 10,000 cells. ( c ) Range of latent infection levels for replicate infections across 3 donors as estimated by CD3/CD28 stimulation of viral reactivation. ( d ) Viral reactivation after 48 hours of stimulation across a panel of LRAs. Data presented as means ± SD (n = 3) for donor 2. ( e , f ) Heatmaps depict phospho-protein time courses following CD3/CD28 ( e ) or PMA/I stimulation ( f ) in uninfected and infected CD4+ T CM cells for 3 donors over 48 hours measured by bead-based immunoassay. Phospho-signals were measured in biological triplicate and 90% of measurements had an SD

    Article Snippet: For primary cells, viral activation was measured by intracellular staining for p24, an HIV-1 capsid protein, with the monoclonal antibody AG3.0 at a 1:40 dilution (NIH AIDS Reagent Program, courtesy of Dr. Jonathan Allan) and anti-mouse IgG secondary antibodies conjugated to AlexaFluor 647 (ThermoFisher Scientific).

    Techniques: Infection, Activation Assay, In Vitro, Cell Culture, Expressing, Flow Cytometry, Cytometry, Bead-based Assay

    DUXAP10 could inhibit LATS2 and RRAD expression A . DUXAP10 expression levels in cell cytoplasm or nucleus of NSCLC cell lines A549 and H1975 were detected by qPCR. GAPDH was used as a cytosol marker and U1 was used as a nucleus marker. B . RIP with rabbit monoclonal anti-LSD1, rabbit monoclonal anti-EZH2, rabbit monoclonal anti-SUZ12, preimmune IgG, or 10% input from A549 and H1975 cell extracts. RNA levels in immunoprecipitates were detected by qPCR. Expression levels of DUXAP10 RNA are presented as fold enrichment in LSD1 relative to IgG immunoprecipitates. C . RNA pulldown and western blotting assays were performed and the results revealed that DUXAP10 could bind to LSD1. D and E . The qPCR and western blot assay were conducted to detect the levels of LATS2 and RRAD mRNA in A549 and H1975 cells transfected with si-DUXAP10 and results are expressed relative to the corresponding values for control cells. F and G . QPCR and Western blot assays were used to detect the LATS2 and RRAD expression both in mRNA and protein levels in A549 and H1975 cells transfected with si-LSD1. H and I . ChIP–qPCR of LSD1 occupancy and H3K4-2me binding in the LATS2 and RRAD promoter in A549 and H1975 cells, and IgG as a negative control. At 48h after transfection, ChIP–qPCR of LSD1 occupancy and H3K4-2me binding in the LATS2 and RRAD promoter in A549 and H1975 cells treated with si-DUXAP10 or scrambled siRNA. *P

    Journal: Oncotarget

    Article Title: The pseudogene DUXAP10 promotes an aggressive phenotype through binding with LSD1 and repressing LATS2 and RRAD in non small cell lung cancer

    doi: 10.18632/oncotarget.14125

    Figure Lengend Snippet: DUXAP10 could inhibit LATS2 and RRAD expression A . DUXAP10 expression levels in cell cytoplasm or nucleus of NSCLC cell lines A549 and H1975 were detected by qPCR. GAPDH was used as a cytosol marker and U1 was used as a nucleus marker. B . RIP with rabbit monoclonal anti-LSD1, rabbit monoclonal anti-EZH2, rabbit monoclonal anti-SUZ12, preimmune IgG, or 10% input from A549 and H1975 cell extracts. RNA levels in immunoprecipitates were detected by qPCR. Expression levels of DUXAP10 RNA are presented as fold enrichment in LSD1 relative to IgG immunoprecipitates. C . RNA pulldown and western blotting assays were performed and the results revealed that DUXAP10 could bind to LSD1. D and E . The qPCR and western blot assay were conducted to detect the levels of LATS2 and RRAD mRNA in A549 and H1975 cells transfected with si-DUXAP10 and results are expressed relative to the corresponding values for control cells. F and G . QPCR and Western blot assays were used to detect the LATS2 and RRAD expression both in mRNA and protein levels in A549 and H1975 cells transfected with si-LSD1. H and I . ChIP–qPCR of LSD1 occupancy and H3K4-2me binding in the LATS2 and RRAD promoter in A549 and H1975 cells, and IgG as a negative control. At 48h after transfection, ChIP–qPCR of LSD1 occupancy and H3K4-2me binding in the LATS2 and RRAD promoter in A549 and H1975 cells treated with si-DUXAP10 or scrambled siRNA. *P

    Article Snippet: One milligram of protein from A549 and H1975 cell extracts were then mixed with 50 pmol of biotinylated RNA, incubated with 50μL of magnetic beads for 1h at 4°C (Thermo Scientific, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Marker, Western Blot, Transfection, Chromatin Immunoprecipitation, Binding Assay, Negative Control

    DUXAP10 negatively regulates expression of LATS2 and RRAD by rescue assays A, B, C, D . MTT, edu and colony formation assays were used to determine the cell proliferation ability for A549 cells transfected with pCDNA-DUXAP10 and pCDNA-LATS2 and pCDNA-RRAD and co-transfected with pCDNA-DUXAP10 and pCDNA-LATS2 or pCDNA-DUXAP10 and pCDNA-RRAD. E . QPCR analyzed the LATS2 and RRAD mRNA levels in 20 pairs NSCLC tissues and found that there was a significantly negative correlation between DUXAP10 and RRAD or LATS2. Values represent the mean ± s.d. from three independent experiments.

    Journal: Oncotarget

    Article Title: The pseudogene DUXAP10 promotes an aggressive phenotype through binding with LSD1 and repressing LATS2 and RRAD in non small cell lung cancer

    doi: 10.18632/oncotarget.14125

    Figure Lengend Snippet: DUXAP10 negatively regulates expression of LATS2 and RRAD by rescue assays A, B, C, D . MTT, edu and colony formation assays were used to determine the cell proliferation ability for A549 cells transfected with pCDNA-DUXAP10 and pCDNA-LATS2 and pCDNA-RRAD and co-transfected with pCDNA-DUXAP10 and pCDNA-LATS2 or pCDNA-DUXAP10 and pCDNA-RRAD. E . QPCR analyzed the LATS2 and RRAD mRNA levels in 20 pairs NSCLC tissues and found that there was a significantly negative correlation between DUXAP10 and RRAD or LATS2. Values represent the mean ± s.d. from three independent experiments.

    Article Snippet: One milligram of protein from A549 and H1975 cell extracts were then mixed with 50 pmol of biotinylated RNA, incubated with 50μL of magnetic beads for 1h at 4°C (Thermo Scientific, USA).

    Techniques: Expressing, MTT Assay, Transfection, Real-time Polymerase Chain Reaction

    Effects of DUXAP10 on NSCLC cell proliferation in vitro A . DUXAP10 expression levels of NSCLC cell lines (A549, H1975, SPC-A1, H1299, H226 and PC-9), compared with that in normal human bronchial epithelial cells (16HBE). B . A549 and H1975 cells were transfected with si-DUXAP10, PC-9 cells were transfected with pCDNA-DUXAP10. C . MTT assays were performed to determine the cell viability for si-DUXAP10-transfected A549 and H1975, and PC-9 cells transfected with pCDNA-DUXAP10. D, E . Colony-forming assays and EDU staining assays were used to determine the proliferation of si-DUXAP10-transfected A549 and H1975 cells. *P

    Journal: Oncotarget

    Article Title: The pseudogene DUXAP10 promotes an aggressive phenotype through binding with LSD1 and repressing LATS2 and RRAD in non small cell lung cancer

    doi: 10.18632/oncotarget.14125

    Figure Lengend Snippet: Effects of DUXAP10 on NSCLC cell proliferation in vitro A . DUXAP10 expression levels of NSCLC cell lines (A549, H1975, SPC-A1, H1299, H226 and PC-9), compared with that in normal human bronchial epithelial cells (16HBE). B . A549 and H1975 cells were transfected with si-DUXAP10, PC-9 cells were transfected with pCDNA-DUXAP10. C . MTT assays were performed to determine the cell viability for si-DUXAP10-transfected A549 and H1975, and PC-9 cells transfected with pCDNA-DUXAP10. D, E . Colony-forming assays and EDU staining assays were used to determine the proliferation of si-DUXAP10-transfected A549 and H1975 cells. *P

    Article Snippet: One milligram of protein from A549 and H1975 cell extracts were then mixed with 50 pmol of biotinylated RNA, incubated with 50μL of magnetic beads for 1h at 4°C (Thermo Scientific, USA).

    Techniques: In Vitro, Expressing, Transfection, MTT Assay, Staining

    The stable DUXAP10 knockdown A549 cells were used for the in vivo study A and B . The nude mice carrying tumors from respective groups were shown and tumor growth curves were measured after the injection of A549 cells. Tumor volume was calculated every 4 days. C . Tumor weights are represented as means of tumor weights ±S.D. D . qPCR assay was performed to determine the average expression of DUXAP10 in xenograft tumors. E . Tumors developed from sh-DUXAP10 transfected A549 cells showed lower Ki67 protein levels than tumors developed by control cells. Upper: H E staining; Lower: immunostaining.

    Journal: Oncotarget

    Article Title: The pseudogene DUXAP10 promotes an aggressive phenotype through binding with LSD1 and repressing LATS2 and RRAD in non small cell lung cancer

    doi: 10.18632/oncotarget.14125

    Figure Lengend Snippet: The stable DUXAP10 knockdown A549 cells were used for the in vivo study A and B . The nude mice carrying tumors from respective groups were shown and tumor growth curves were measured after the injection of A549 cells. Tumor volume was calculated every 4 days. C . Tumor weights are represented as means of tumor weights ±S.D. D . qPCR assay was performed to determine the average expression of DUXAP10 in xenograft tumors. E . Tumors developed from sh-DUXAP10 transfected A549 cells showed lower Ki67 protein levels than tumors developed by control cells. Upper: H E staining; Lower: immunostaining.

    Article Snippet: One milligram of protein from A549 and H1975 cell extracts were then mixed with 50 pmol of biotinylated RNA, incubated with 50μL of magnetic beads for 1h at 4°C (Thermo Scientific, USA).

    Techniques: In Vivo, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Expressing, Transfection, Staining, Immunostaining

    Effect of LATS2 and RRAD of overexpression on A549 cell in vitro A, B . The mRNA levels and protein levels of LATS2 and RRAD in A549 cells transfected with pCDNA–LATS2 or pCDNA-RRAD was detected by qPCR analysis. C, D . MTT assays and Edu staining assays were used to determine the cell viability. Values represent the mean ± s.d. from three independent experiments. E . Cell cycle was analyzed by flow cytometry. The bar chart represents the percentage of cells in G1–G0, S, or G2–M phase, as indicated. *P

    Journal: Oncotarget

    Article Title: The pseudogene DUXAP10 promotes an aggressive phenotype through binding with LSD1 and repressing LATS2 and RRAD in non small cell lung cancer

    doi: 10.18632/oncotarget.14125

    Figure Lengend Snippet: Effect of LATS2 and RRAD of overexpression on A549 cell in vitro A, B . The mRNA levels and protein levels of LATS2 and RRAD in A549 cells transfected with pCDNA–LATS2 or pCDNA-RRAD was detected by qPCR analysis. C, D . MTT assays and Edu staining assays were used to determine the cell viability. Values represent the mean ± s.d. from three independent experiments. E . Cell cycle was analyzed by flow cytometry. The bar chart represents the percentage of cells in G1–G0, S, or G2–M phase, as indicated. *P

    Article Snippet: One milligram of protein from A549 and H1975 cell extracts were then mixed with 50 pmol of biotinylated RNA, incubated with 50μL of magnetic beads for 1h at 4°C (Thermo Scientific, USA).

    Techniques: Over Expression, In Vitro, Transfection, Real-time Polymerase Chain Reaction, MTT Assay, Staining, Flow Cytometry, Cytometry

    miR-21 is poorly expressed in BALF of PGD patient where NET is a contributor for PGD. a, miR-21 was poorly expressed in BALF of PGD patients determined by RT-qPCR; b, miR-21 expression was conversely correlated to PGD grades, as detected by RT-qPCR; c, the expression of IL-6, CXCL10, CCL2, and IL-8 was higher in BALF of PGD than that in non-PGD patients, as measured by ELISA; d, the cell population of leukocytes was increased whilst that of macrophages was diminished in BALF of PGD patients; e, the level of free DNA was promoted in BALF of PGD patients, as detected by immunofluorescence staining; f, miR-21 expressed at a low level in BAL cells. All data were presented as mean ± standard deviation. The data between two groups were compared and analysed using unpaired t -test; n = 18 (non-PGD patients); n = 24 (PGD patients); * p

    Journal: EBioMedicine

    Article Title: Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism

    doi: 10.1016/j.ebiom.2019.102600

    Figure Lengend Snippet: miR-21 is poorly expressed in BALF of PGD patient where NET is a contributor for PGD. a, miR-21 was poorly expressed in BALF of PGD patients determined by RT-qPCR; b, miR-21 expression was conversely correlated to PGD grades, as detected by RT-qPCR; c, the expression of IL-6, CXCL10, CCL2, and IL-8 was higher in BALF of PGD than that in non-PGD patients, as measured by ELISA; d, the cell population of leukocytes was increased whilst that of macrophages was diminished in BALF of PGD patients; e, the level of free DNA was promoted in BALF of PGD patients, as detected by immunofluorescence staining; f, miR-21 expressed at a low level in BAL cells. All data were presented as mean ± standard deviation. The data between two groups were compared and analysed using unpaired t -test; n = 18 (non-PGD patients); n = 24 (PGD patients); * p

    Article Snippet: 2.11 Reverse transcription quantitative polymerase chain reaction (RT-qPCR) The total RNA was extracted from PMNs or rat BALF using the Trizol kit (16,096,020, Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Standard Deviation

    LncRNA XIST upregulates IL-12A by binding to miR-21. a, there were binding sites between miR-21 and XIST, according to online prediction; b, the luciferase activity of XIST-wt was inhibited by miR-21 mimic, as confirmed by the dual luciferase reporter assay; c, XIST was pulled-down by miR-21-Mut in RNA pull-down assay; d, the expression of miR-21 and XIST coprecipitated with Ago2 magnetic beads was enhanced in the RIP assay; e, XIST was upregulated in BALF of PGD patients, as measured by RT-qPCR; f, the expression of miR-21 was promoted and IL-12A expression was suppressed in PMNs after the suppression of XIST, as measured by RT-qPCR; g, there was a negative correlation between miR-21 and XIST, as determined by the correlation analysis; h, IL-12A and XIST were positively correlated, as determined by the correlation analysis. All data were presented as mean ± standard deviation. The data between PGD patients ( n = 24) and non-PGD patients ( n = 18) in panel e were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; * p

    Journal: EBioMedicine

    Article Title: Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism

    doi: 10.1016/j.ebiom.2019.102600

    Figure Lengend Snippet: LncRNA XIST upregulates IL-12A by binding to miR-21. a, there were binding sites between miR-21 and XIST, according to online prediction; b, the luciferase activity of XIST-wt was inhibited by miR-21 mimic, as confirmed by the dual luciferase reporter assay; c, XIST was pulled-down by miR-21-Mut in RNA pull-down assay; d, the expression of miR-21 and XIST coprecipitated with Ago2 magnetic beads was enhanced in the RIP assay; e, XIST was upregulated in BALF of PGD patients, as measured by RT-qPCR; f, the expression of miR-21 was promoted and IL-12A expression was suppressed in PMNs after the suppression of XIST, as measured by RT-qPCR; g, there was a negative correlation between miR-21 and XIST, as determined by the correlation analysis; h, IL-12A and XIST were positively correlated, as determined by the correlation analysis. All data were presented as mean ± standard deviation. The data between PGD patients ( n = 24) and non-PGD patients ( n = 18) in panel e were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; * p

    Article Snippet: 2.11 Reverse transcription quantitative polymerase chain reaction (RT-qPCR) The total RNA was extracted from PMNs or rat BALF using the Trizol kit (16,096,020, Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Binding Assay, Luciferase, Activity Assay, Reporter Assay, Pull Down Assay, Expressing, Magnetic Beads, Quantitative RT-PCR, Standard Deviation

    IL-12A is a target gene of miR-21, and can reverse the inhibitory effect of miR-21 on NET formation. a, there were binding sites between miR-21 and IL-12A, as predicted on the TargetScan software ( http://www.targetscan.org/ ); b, IL-12A-wt expression was repressed by miR-21 mimic verified by the dual luciferase reporter assay; c, the mRNA expression of IL-12A in PMNs following transfection with miR-21 mimic was inhibited, as detected by RT-qPCR; d, the protein expression of IL-12A was suppressed in response to the treatment with miR-21 mimic, as determined by western blot analysis; e, the protein expression of IL-12A in BALF of PGD patients was enhanced, as determined by western blot analysis; f, the mRNA expression of IL-12A in BALF of PGD patients was facilitated, as determined by RT-qPCR; g, the content of NET-DNA in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic was reduced, as assessed by ELISA; h, the apoptosis rate was promoted in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by flow cytometry; i, the mRNA expression of IL-12A in BALF of rats was diminished after miR-21 mimic treatment, as determined by RT-qPCR; j, the content of free DNA was decreased in PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by immunofluorescence staining (400 ×). All data were presented as mean ± standard deviation. The data between two groups were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; in panel f, n = 18 (non-PGD patients); n = 24 (PGD patients); * p

    Journal: EBioMedicine

    Article Title: Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism

    doi: 10.1016/j.ebiom.2019.102600

    Figure Lengend Snippet: IL-12A is a target gene of miR-21, and can reverse the inhibitory effect of miR-21 on NET formation. a, there were binding sites between miR-21 and IL-12A, as predicted on the TargetScan software ( http://www.targetscan.org/ ); b, IL-12A-wt expression was repressed by miR-21 mimic verified by the dual luciferase reporter assay; c, the mRNA expression of IL-12A in PMNs following transfection with miR-21 mimic was inhibited, as detected by RT-qPCR; d, the protein expression of IL-12A was suppressed in response to the treatment with miR-21 mimic, as determined by western blot analysis; e, the protein expression of IL-12A in BALF of PGD patients was enhanced, as determined by western blot analysis; f, the mRNA expression of IL-12A in BALF of PGD patients was facilitated, as determined by RT-qPCR; g, the content of NET-DNA in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic was reduced, as assessed by ELISA; h, the apoptosis rate was promoted in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by flow cytometry; i, the mRNA expression of IL-12A in BALF of rats was diminished after miR-21 mimic treatment, as determined by RT-qPCR; j, the content of free DNA was decreased in PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by immunofluorescence staining (400 ×). All data were presented as mean ± standard deviation. The data between two groups were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; in panel f, n = 18 (non-PGD patients); n = 24 (PGD patients); * p

    Article Snippet: 2.11 Reverse transcription quantitative polymerase chain reaction (RT-qPCR) The total RNA was extracted from PMNs or rat BALF using the Trizol kit (16,096,020, Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Binding Assay, Software, Expressing, Luciferase, Reporter Assay, Transfection, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunofluorescence, Staining, Standard Deviation

    Antitumor activity of SASP from Stat3-silenced cancer cells. 4T1, B16, MDA-MB231, and MCA101 cells were transfected with 100 nmol/L siControl or siStat3 and conditioned mediums (CM-siControl or CM-siStat3, respectively) were obtained as described in Materials and methods. (a) Tumor cells were cultured for 48 h with CM-siControl or CM-siStat3 produced by the same cell line as indicated. Proliferation was determined by [ 3 H] thymidine incorporation (n = 8). (b) Monolayers of tumor cells were wounded and allowed to migrate into the cell-free area in presence of CM-siControl or CM-siStat3 of tumor cells or control medium as indicated. Wounded areas were photographed at 0 and 18 h and quantified by densitometry (n = 4). (c) HUVEC cells were cultured for 48 h in presence of CM-siControl or CM-siStat3 of tumor cells or control medium as indicated, and proliferation was measured by [ 3 H] thymidine incorporation (n = 8). (d-f) Purified T cells from spleen of BALB/c mice were activated with CD3/CD28 beads in the presence of CM-siControl or CM-siStat3 from 4T1 or MCA101 cells for 72 h. Similar protocols were followed with CM of B16 cells but T cells were isolated from the spleen of C57BL/6 mice. (d) Proliferation was determined by [ 3 H] thymidine incorporation at 48 h (n = 8). (e) Activation of CD4+ or CD8 + T cells was determined immunofluorescence using anti-CD69 antibody and flow cytometry (n = 4). (f) Determination of CD4 + T and CD8 + T effector, central memory, and naïve T cells were determined by immunofluorescence using anti-CD62L and CD44 antibodies, and flow cytometry (n = 4). Data are presented as means ± SE. All data shown are representative of three independent experiments. p values were calculated using two-tailed Student´s t test in a, b, d–f and by one-way ANOVA with Tukey’s post-test in c . ns, not significant; * p

    Journal: Oncoimmunology

    Article Title: Blockade of Stat3 oncogene addiction induces cellular senescence and reveals a cell-nonautonomous activity suitable for cancer immunotherapy

    doi: 10.1080/2162402X.2020.1715767

    Figure Lengend Snippet: Antitumor activity of SASP from Stat3-silenced cancer cells. 4T1, B16, MDA-MB231, and MCA101 cells were transfected with 100 nmol/L siControl or siStat3 and conditioned mediums (CM-siControl or CM-siStat3, respectively) were obtained as described in Materials and methods. (a) Tumor cells were cultured for 48 h with CM-siControl or CM-siStat3 produced by the same cell line as indicated. Proliferation was determined by [ 3 H] thymidine incorporation (n = 8). (b) Monolayers of tumor cells were wounded and allowed to migrate into the cell-free area in presence of CM-siControl or CM-siStat3 of tumor cells or control medium as indicated. Wounded areas were photographed at 0 and 18 h and quantified by densitometry (n = 4). (c) HUVEC cells were cultured for 48 h in presence of CM-siControl or CM-siStat3 of tumor cells or control medium as indicated, and proliferation was measured by [ 3 H] thymidine incorporation (n = 8). (d-f) Purified T cells from spleen of BALB/c mice were activated with CD3/CD28 beads in the presence of CM-siControl or CM-siStat3 from 4T1 or MCA101 cells for 72 h. Similar protocols were followed with CM of B16 cells but T cells were isolated from the spleen of C57BL/6 mice. (d) Proliferation was determined by [ 3 H] thymidine incorporation at 48 h (n = 8). (e) Activation of CD4+ or CD8 + T cells was determined immunofluorescence using anti-CD69 antibody and flow cytometry (n = 4). (f) Determination of CD4 + T and CD8 + T effector, central memory, and naïve T cells were determined by immunofluorescence using anti-CD62L and CD44 antibodies, and flow cytometry (n = 4). Data are presented as means ± SE. All data shown are representative of three independent experiments. p values were calculated using two-tailed Student´s t test in a, b, d–f and by one-way ANOVA with Tukey’s post-test in c . ns, not significant; * p

    Article Snippet: For proliferation assay, stimulation with CD3/CD28 beads was performed for 72 h in 96-well plates using a 0.5:1 beads:lymphocyte ratio.

    Techniques: Activity Assay, Multiple Displacement Amplification, Transfection, Cell Culture, Produced, Purification, Mouse Assay, Isolation, Activation Assay, Immunofluorescence, Flow Cytometry, Two Tailed Test

    Senescence and type I IFN-related protein production induced by Stat3 silencing is dependent on cGAS. 4T1 and B16 cells were transfected with siControl, siStat3, or cGAS siRNA (sicGAS) or with siStat3+ sicGAS siRNA for 48 h. (a) Senescence was determined by labeling with 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C 12 FDG). The fluorescence was quantified by flow cytometry thought the mean fluorescence intensity (MFI). (b) Determination of CXCL10 by ELISA in the CM. (c) Determination ISG15 and IFI35 by immunoblot from cell lysates. (d,e) Purified T cells from spleen of BALB/c mice were activated with CD3/CD28 beads in the presence of CM from 4T1 cells. Similar protocols were followed with CM of B16 cell but T cells were isolated from the spleen of C57BL/6 mice. (d) Proliferation was determined by [ 3 H] thymidine incorporation at 48 h (n = 8). (e) Proliferation was determined by [ 3 H] thymidine incorporation at 48 h (n = 8). In the case of CM-siStat3 anti-IFNAR antibody was added. (f) Immunotherapy protocol. BALB/c mice were challenged s.c. with 10 4 4T1 cells. When tumors were palpable, animals were injected s.c. with 10 6 irradiated 4T1 cells (100 Gy) and with a depot containing the lyophilized CM-siControl or CM-siStat3 or CM-sicGAS or CM-sicGAS+siStat3 from 4T1 cells once a week for 3 weeks (n = 5 mice per group). (g) Tumor volume was monitored along the experiment. Data are presented as means ± SE. Data shown are representative of three independent experiments ( a–e ) and of one experiment ( g ). p values were calculated using ANOVA with Tukey’s posttest in a, b, d, e and two-way ANOVA test in g . * p

    Journal: Oncoimmunology

    Article Title: Blockade of Stat3 oncogene addiction induces cellular senescence and reveals a cell-nonautonomous activity suitable for cancer immunotherapy

    doi: 10.1080/2162402X.2020.1715767

    Figure Lengend Snippet: Senescence and type I IFN-related protein production induced by Stat3 silencing is dependent on cGAS. 4T1 and B16 cells were transfected with siControl, siStat3, or cGAS siRNA (sicGAS) or with siStat3+ sicGAS siRNA for 48 h. (a) Senescence was determined by labeling with 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C 12 FDG). The fluorescence was quantified by flow cytometry thought the mean fluorescence intensity (MFI). (b) Determination of CXCL10 by ELISA in the CM. (c) Determination ISG15 and IFI35 by immunoblot from cell lysates. (d,e) Purified T cells from spleen of BALB/c mice were activated with CD3/CD28 beads in the presence of CM from 4T1 cells. Similar protocols were followed with CM of B16 cell but T cells were isolated from the spleen of C57BL/6 mice. (d) Proliferation was determined by [ 3 H] thymidine incorporation at 48 h (n = 8). (e) Proliferation was determined by [ 3 H] thymidine incorporation at 48 h (n = 8). In the case of CM-siStat3 anti-IFNAR antibody was added. (f) Immunotherapy protocol. BALB/c mice were challenged s.c. with 10 4 4T1 cells. When tumors were palpable, animals were injected s.c. with 10 6 irradiated 4T1 cells (100 Gy) and with a depot containing the lyophilized CM-siControl or CM-siStat3 or CM-sicGAS or CM-sicGAS+siStat3 from 4T1 cells once a week for 3 weeks (n = 5 mice per group). (g) Tumor volume was monitored along the experiment. Data are presented as means ± SE. Data shown are representative of three independent experiments ( a–e ) and of one experiment ( g ). p values were calculated using ANOVA with Tukey’s posttest in a, b, d, e and two-way ANOVA test in g . * p

    Article Snippet: For proliferation assay, stimulation with CD3/CD28 beads was performed for 72 h in 96-well plates using a 0.5:1 beads:lymphocyte ratio.

    Techniques: Transfection, Labeling, Fluorescence, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Purification, Mouse Assay, Isolation, Injection, Irradiation

    CCL2, CCL5, IL-15, and CXCL10 are mediators of the immune-stimulating activity of the SASP from Stat3-silenced cancer cells. (a) CM-siControl and CM-siStat3 from 4T1 and B16 cells were analyzed using an antibody array for detecting cytokine and chemokine secretion. Heatmap of differentially expressed proteins in CM-siStat3 vs. CM-siControl. (b,c) Determination of different cytokines and chemokines in CM-siControl and CM-siStat3 from 4T1, B16, and MCA101 cells by ELISA. (d) Effect of neutralization of CXCR3, CCL2, CCL5, and IL-15 with specific antibodies on T cell proliferation. Purified T cells from spleen of BALB/c mice were activated with CD3/CD28 beads in the presence of CM-siControl or CM-siStat3 from 4T1 cells for 48 h and the corresponding Armenian hamster or goat control antibodies. In the case of CM-siStat3 different blocking antibodies were included. Data shown of CM-siControl and CM-siStat3 correspond to that obtained with Armenian hamster antibody, yielding similar results to those from goat antibody (not shown). Similar protocols were followed with CM from B16 cells but T cells were isolated from spleen of C57BL/6 mice. Proliferation was determined by [ 3 H] thymidine incorporation at 72 h. Data are presented as means ± SE. Data shown are representative of two ( a, b ), or three ( c, d ), independent experiments. p values were calculated using two-tailed Student´s t test in b, c and by ANOVA with Tukey’s posttest in d . ns, not significant, * p

    Journal: Oncoimmunology

    Article Title: Blockade of Stat3 oncogene addiction induces cellular senescence and reveals a cell-nonautonomous activity suitable for cancer immunotherapy

    doi: 10.1080/2162402X.2020.1715767

    Figure Lengend Snippet: CCL2, CCL5, IL-15, and CXCL10 are mediators of the immune-stimulating activity of the SASP from Stat3-silenced cancer cells. (a) CM-siControl and CM-siStat3 from 4T1 and B16 cells were analyzed using an antibody array for detecting cytokine and chemokine secretion. Heatmap of differentially expressed proteins in CM-siStat3 vs. CM-siControl. (b,c) Determination of different cytokines and chemokines in CM-siControl and CM-siStat3 from 4T1, B16, and MCA101 cells by ELISA. (d) Effect of neutralization of CXCR3, CCL2, CCL5, and IL-15 with specific antibodies on T cell proliferation. Purified T cells from spleen of BALB/c mice were activated with CD3/CD28 beads in the presence of CM-siControl or CM-siStat3 from 4T1 cells for 48 h and the corresponding Armenian hamster or goat control antibodies. In the case of CM-siStat3 different blocking antibodies were included. Data shown of CM-siControl and CM-siStat3 correspond to that obtained with Armenian hamster antibody, yielding similar results to those from goat antibody (not shown). Similar protocols were followed with CM from B16 cells but T cells were isolated from spleen of C57BL/6 mice. Proliferation was determined by [ 3 H] thymidine incorporation at 72 h. Data are presented as means ± SE. Data shown are representative of two ( a, b ), or three ( c, d ), independent experiments. p values were calculated using two-tailed Student´s t test in b, c and by ANOVA with Tukey’s posttest in d . ns, not significant, * p

    Article Snippet: For proliferation assay, stimulation with CD3/CD28 beads was performed for 72 h in 96-well plates using a 0.5:1 beads:lymphocyte ratio.

    Techniques: Activity Assay, Ab Array, Enzyme-linked Immunosorbent Assay, Neutralization, Purification, Mouse Assay, Blocking Assay, Isolation, Two Tailed Test