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  • 99
    Millipore ficoll density gradient centrifugation
    Rainbow trout RBC transformation sequence to shRBC induced by temperature increases. <t>Ficoll-purified</t> <t>RBCs</t> were incubated for 3 days at different temperatures: ( a ) 14 °C, ( b ) 25 °C and ( c ) 28 °C. The black arrow indicate shRBCs. Representative bright-field microscopy images of the sequence of morphological changes in RBCs were taken with 60× magnification: ( d ) RBC, ( e ) stressed RBC, ( f ) round RBC with extruding nucleus and ( g ) shRBC.
    Ficoll Density Gradient Centrifugation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 957 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare ficoll hypaque density gradient centrifugation
    Double hit model with LPS. Figure 1A . Diagram of the model of endotoxin tolerance used in our study. Peripheral blood mononuclear cells (PBMCs) were obtained from fresh whole blood from healthy volunteers (HV) by <t>Ficoll-Paque</t> density gradient centrifugation. Endotoxin tolerance was reproduced ex vivo by priming PBMCs with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. Figure 1B . Diagram of immune-stimulating therapy model during endotoxin tolerance.
    Ficoll Hypaque Density Gradient Centrifugation, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ficoll hypaque density gradient centrifugation
    Double hit model with LPS. Figure 1A . Diagram of the model of endotoxin tolerance used in our study. Peripheral blood mononuclear cells (PBMCs) were obtained from fresh whole blood from healthy volunteers (HV) by <t>Ficoll-Paque</t> density gradient centrifugation. Endotoxin tolerance was reproduced ex vivo by priming PBMCs with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. Figure 1B . Diagram of immune-stimulating therapy model during endotoxin tolerance.
    Ficoll Hypaque Density Gradient Centrifugation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 884 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Ficoll-Paque Pharmacia density gradient centrifugation
    Double hit model with LPS. Figure 1A . Diagram of the model of endotoxin tolerance used in our study. Peripheral blood mononuclear cells (PBMCs) were obtained from fresh whole blood from healthy volunteers (HV) by <t>Ficoll-Paque</t> density gradient centrifugation. Endotoxin tolerance was reproduced ex vivo by priming PBMCs with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. Figure 1B . Diagram of immune-stimulating therapy model during endotoxin tolerance.
    Density Gradient Centrifugation, supplied by Ficoll-Paque Pharmacia, used in various techniques. Bioz Stars score: 92/100, based on 147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biochrom ficoll density gradient centrifugation
    Dot blot kinetics of MDSC. Representative dot plots of gMDSC ( a–c ) and mMDSC ( d–f ) after 2, 4, 6 h, overnight rest and freezing/thawing procedure of three HIV patients (gMDSC) and three patients with solid tumors (mMDSC). <t>PBMC</t> were stored in RPMI media after the <t>ficoll</t> separation gMDSC gated on CD11b + and CD14 − population ( oval gate); for CD15 + /CD66b + gating see Additional file 2 : Figure S2; mMDSC gated on CD33 + and HLA-DR low/− ( rectangle gate); CD14 + gating not shown. The majority of dot blots ( a ) of the CD11b + /CD14 − population remained similar after 2, 4 and 6 h. However, it was not clearly definable after ON and almost disappeared after the freezing/thawing procedure. Divergent minor presentations were seen and are presented in the remaining panels: increasing gMDSC frequencies after overnight rest and relatively high gMDSC levels after freezing/thawing ( b ) as well as reduction of gMDSC numbers at 4, 6 h and overnight rest compared to the 2 h time point ( c ). Looking at mMDSC (CD33 + and HLADR −/low population), again the majority of dot blots presented as shown in 2d: similar numbers of mMDSC after 2 and 4 h and a clear reduction in mMDSC frequencies after 6 h. Populations are not well discriminable after overnight rest and after freezing/thawing. Minor divergent presentations are shown in the remaining panels: very high mMDSC frequencies after freezing/thawing ( e ) as well as increased frequencies after overnight rest and loss of mMDSC after freezing/thawing ( f )
    Ficoll Density Gradient Centrifugation, supplied by Biochrom, used in various techniques. Bioz Stars score: 92/100, based on 333 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore density gradient centrifugation
    Dot blot kinetics of MDSC. Representative dot plots of gMDSC ( a–c ) and mMDSC ( d–f ) after 2, 4, 6 h, overnight rest and freezing/thawing procedure of three HIV patients (gMDSC) and three patients with solid tumors (mMDSC). <t>PBMC</t> were stored in RPMI media after the <t>ficoll</t> separation gMDSC gated on CD11b + and CD14 − population ( oval gate); for CD15 + /CD66b + gating see Additional file 2 : Figure S2; mMDSC gated on CD33 + and HLA-DR low/− ( rectangle gate); CD14 + gating not shown. The majority of dot blots ( a ) of the CD11b + /CD14 − population remained similar after 2, 4 and 6 h. However, it was not clearly definable after ON and almost disappeared after the freezing/thawing procedure. Divergent minor presentations were seen and are presented in the remaining panels: increasing gMDSC frequencies after overnight rest and relatively high gMDSC levels after freezing/thawing ( b ) as well as reduction of gMDSC numbers at 4, 6 h and overnight rest compared to the 2 h time point ( c ). Looking at mMDSC (CD33 + and HLADR −/low population), again the majority of dot blots presented as shown in 2d: similar numbers of mMDSC after 2 and 4 h and a clear reduction in mMDSC frequencies after 6 h. Populations are not well discriminable after overnight rest and after freezing/thawing. Minor divergent presentations are shown in the remaining panels: very high mMDSC frequencies after freezing/thawing ( e ) as well as increased frequencies after overnight rest and loss of mMDSC after freezing/thawing ( f )
    Density Gradient Centrifugation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 992 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare percoll density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Percoll Density Gradient Centrifugation, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Axis-Shield Diagnostics ficoll hypaque density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Ficoll Hypaque Density Gradient Centrifugation, supplied by Axis-Shield Diagnostics, used in various techniques. Bioz Stars score: 92/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson ficoll density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Ficoll Density Gradient Centrifugation, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biochrom ficoll hypaque density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Ficoll Hypaque Density Gradient Centrifugation, supplied by Biochrom, used in various techniques. Bioz Stars score: 93/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore percoll density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Percoll Density Gradient Centrifugation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Eurobio ficoll density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Ficoll Density Gradient Centrifugation, supplied by Eurobio, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axis-Shield Diagnostics lymphoprep density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Lymphoprep Density Gradient Centrifugation, supplied by Axis-Shield Diagnostics, used in various techniques. Bioz Stars score: 89/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axis-Shield Diagnostics density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Density Gradient Centrifugation, supplied by Axis-Shield Diagnostics, used in various techniques. Bioz Stars score: 93/100, based on 318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore histopaque sigma aldrich density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Histopaque Sigma Aldrich Density Gradient Centrifugation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ficoll histopaque density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Ficoll Histopaque Density Gradient Centrifugation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nycomed ficoll density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
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    89
    Thermo Fisher ficoll isopaque density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
    Ficoll Isopaque Density Gradient Centrifugation, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biochrom biocoll density gradient centrifugation
    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by <t>Percoll</t> gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.
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    Image Search Results


    Rainbow trout RBC transformation sequence to shRBC induced by temperature increases. Ficoll-purified RBCs were incubated for 3 days at different temperatures: ( a ) 14 °C, ( b ) 25 °C and ( c ) 28 °C. The black arrow indicate shRBCs. Representative bright-field microscopy images of the sequence of morphological changes in RBCs were taken with 60× magnification: ( d ) RBC, ( e ) stressed RBC, ( f ) round RBC with extruding nucleus and ( g ) shRBC.

    Journal: Cells

    Article Title: Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

    doi: 10.3390/cells7040031

    Figure Lengend Snippet: Rainbow trout RBC transformation sequence to shRBC induced by temperature increases. Ficoll-purified RBCs were incubated for 3 days at different temperatures: ( a ) 14 °C, ( b ) 25 °C and ( c ) 28 °C. The black arrow indicate shRBCs. Representative bright-field microscopy images of the sequence of morphological changes in RBCs were taken with 60× magnification: ( d ) RBC, ( e ) stressed RBC, ( f ) round RBC with extruding nucleus and ( g ) shRBC.

    Article Snippet: Briefly, RBCs obtained from the caudal vein were purified by Ficoll density gradient centrifugation (Ficoll 1.007; Sigma-Aldrich, Madrid, Spain).

    Techniques: Transformation Assay, Sequencing, Purification, Incubation, Microscopy

    Phosphatidylserine expression on the membrane of RBCs and shRBCs. Ficoll-purified RBCs and shRBCs were obtained after incubation of RBCs at 14 °C, 25 °C and 28 °C, for 6 days. ( a ) Representative fluorescence microscopy images stained with annexin V and DAPI. ( b ) Mean fluorescence intensity (MFI) of annexin staining levels by flow cytometry. Data represent mean ± SD (n = 4). Kruskal–Wallis Test with Dunn’s Multiple Comparison post-hoc test was performed among all conditions. ** p -value

    Journal: Cells

    Article Title: Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

    doi: 10.3390/cells7040031

    Figure Lengend Snippet: Phosphatidylserine expression on the membrane of RBCs and shRBCs. Ficoll-purified RBCs and shRBCs were obtained after incubation of RBCs at 14 °C, 25 °C and 28 °C, for 6 days. ( a ) Representative fluorescence microscopy images stained with annexin V and DAPI. ( b ) Mean fluorescence intensity (MFI) of annexin staining levels by flow cytometry. Data represent mean ± SD (n = 4). Kruskal–Wallis Test with Dunn’s Multiple Comparison post-hoc test was performed among all conditions. ** p -value

    Article Snippet: Briefly, RBCs obtained from the caudal vein were purified by Ficoll density gradient centrifugation (Ficoll 1.007; Sigma-Aldrich, Madrid, Spain).

    Techniques: Expressing, Purification, Incubation, Fluorescence, Microscopy, Staining, Flow Cytometry, Cytometry

    Characterization of rainbow trout RBC transformation to shRBC. Ficoll-purified RBCs were incubated at 25 °C for 3 days. ( a ) Representative images of Wright–Giemsa staining were taken with 40× magnification and are shown, along with TEM micrographs of ( b ) characteristic RBCs, ( c ) stressed RBCs and ( d , e ) shRBCs. Black arrowhead: RBC; black star: shRBC. TEM images were taken with: ( b ) 5k×, ( c ) 8k×, ( d ) 10k×, and ( e ) 50k× magnifications.

    Journal: Cells

    Article Title: Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

    doi: 10.3390/cells7040031

    Figure Lengend Snippet: Characterization of rainbow trout RBC transformation to shRBC. Ficoll-purified RBCs were incubated at 25 °C for 3 days. ( a ) Representative images of Wright–Giemsa staining were taken with 40× magnification and are shown, along with TEM micrographs of ( b ) characteristic RBCs, ( c ) stressed RBCs and ( d , e ) shRBCs. Black arrowhead: RBC; black star: shRBC. TEM images were taken with: ( b ) 5k×, ( c ) 8k×, ( d ) 10k×, and ( e ) 50k× magnifications.

    Article Snippet: Briefly, RBCs obtained from the caudal vein were purified by Ficoll density gradient centrifugation (Ficoll 1.007; Sigma-Aldrich, Madrid, Spain).

    Techniques: Transformation Assay, Purification, Incubation, Staining, Transmission Electron Microscopy

    Double hit model with LPS. Figure 1A . Diagram of the model of endotoxin tolerance used in our study. Peripheral blood mononuclear cells (PBMCs) were obtained from fresh whole blood from healthy volunteers (HV) by Ficoll-Paque density gradient centrifugation. Endotoxin tolerance was reproduced ex vivo by priming PBMCs with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. Figure 1B . Diagram of immune-stimulating therapy model during endotoxin tolerance.

    Journal: PLoS ONE

    Article Title: S100A8/A9 mRNA Induction in an Ex Vivo Model of Endotoxin Tolerance: Roles of IL-10 and IFN?

    doi: 10.1371/journal.pone.0100909

    Figure Lengend Snippet: Double hit model with LPS. Figure 1A . Diagram of the model of endotoxin tolerance used in our study. Peripheral blood mononuclear cells (PBMCs) were obtained from fresh whole blood from healthy volunteers (HV) by Ficoll-Paque density gradient centrifugation. Endotoxin tolerance was reproduced ex vivo by priming PBMCs with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. Figure 1B . Diagram of immune-stimulating therapy model during endotoxin tolerance.

    Article Snippet: Isolation of PBMCs and cell culture conditions PBMCs (peripheral blood mononuclear cells) were obtained from fresh whole blood by Ficoll-Paque density gradient centrifugation (GE Healthcare, Uppsala, Sweden) and washed with sterile PBS (phosphate buffered saline) (Invitrogen, Carlsbad, CA, USA).

    Techniques: Gradient Centrifugation, Ex Vivo

    Dot blot kinetics of MDSC. Representative dot plots of gMDSC ( a–c ) and mMDSC ( d–f ) after 2, 4, 6 h, overnight rest and freezing/thawing procedure of three HIV patients (gMDSC) and three patients with solid tumors (mMDSC). PBMC were stored in RPMI media after the ficoll separation gMDSC gated on CD11b + and CD14 − population ( oval gate); for CD15 + /CD66b + gating see Additional file 2 : Figure S2; mMDSC gated on CD33 + and HLA-DR low/− ( rectangle gate); CD14 + gating not shown. The majority of dot blots ( a ) of the CD11b + /CD14 − population remained similar after 2, 4 and 6 h. However, it was not clearly definable after ON and almost disappeared after the freezing/thawing procedure. Divergent minor presentations were seen and are presented in the remaining panels: increasing gMDSC frequencies after overnight rest and relatively high gMDSC levels after freezing/thawing ( b ) as well as reduction of gMDSC numbers at 4, 6 h and overnight rest compared to the 2 h time point ( c ). Looking at mMDSC (CD33 + and HLADR −/low population), again the majority of dot blots presented as shown in 2d: similar numbers of mMDSC after 2 and 4 h and a clear reduction in mMDSC frequencies after 6 h. Populations are not well discriminable after overnight rest and after freezing/thawing. Minor divergent presentations are shown in the remaining panels: very high mMDSC frequencies after freezing/thawing ( e ) as well as increased frequencies after overnight rest and loss of mMDSC after freezing/thawing ( f )

    Journal: Journal of Translational Medicine

    Article Title: Kinetics of human myeloid-derived suppressor cells after blood draw

    doi: 10.1186/s12967-015-0755-y

    Figure Lengend Snippet: Dot blot kinetics of MDSC. Representative dot plots of gMDSC ( a–c ) and mMDSC ( d–f ) after 2, 4, 6 h, overnight rest and freezing/thawing procedure of three HIV patients (gMDSC) and three patients with solid tumors (mMDSC). PBMC were stored in RPMI media after the ficoll separation gMDSC gated on CD11b + and CD14 − population ( oval gate); for CD15 + /CD66b + gating see Additional file 2 : Figure S2; mMDSC gated on CD33 + and HLA-DR low/− ( rectangle gate); CD14 + gating not shown. The majority of dot blots ( a ) of the CD11b + /CD14 − population remained similar after 2, 4 and 6 h. However, it was not clearly definable after ON and almost disappeared after the freezing/thawing procedure. Divergent minor presentations were seen and are presented in the remaining panels: increasing gMDSC frequencies after overnight rest and relatively high gMDSC levels after freezing/thawing ( b ) as well as reduction of gMDSC numbers at 4, 6 h and overnight rest compared to the 2 h time point ( c ). Looking at mMDSC (CD33 + and HLADR −/low population), again the majority of dot blots presented as shown in 2d: similar numbers of mMDSC after 2 and 4 h and a clear reduction in mMDSC frequencies after 6 h. Populations are not well discriminable after overnight rest and after freezing/thawing. Minor divergent presentations are shown in the remaining panels: very high mMDSC frequencies after freezing/thawing ( e ) as well as increased frequencies after overnight rest and loss of mMDSC after freezing/thawing ( f )

    Article Snippet: PBMC isolation, freezing and thawing PBMC were isolated from freshly obtained EDTA blood by Ficoll density gradient centrifugation (Biocoll Separation Solution, Biochrom, Germany).

    Techniques: Dot Blot

    Kinetics of gMDSC and mMDSC levels. Frequency of gMDSC ( a ) and mMDSC ( b ) within PBMC after 2, 4, 6 h, overnight rest (ON) and freezing/thawing procedure (defrost). PBMC were stored in RPMI media after the ficoll separation. gMDSC were analysed in 24 patients with chronic viral infections and mMDSC in 17 patients with solid tumors and three HIV infected patients. There was no statistical significant difference in gMDSC frequencies over time (2, 4, 6 h, ON). Statistical significant reduction of gMDSC frequencies after freezing/thawing procedure (p = 0.0001; Mann–Whitney test). The difference in frequencies in mMDSC is not significant after 4 h whereas their numbers were significantly reduced after a 6 h and overnight rest (p = 0.0005 and p = 0.005, respectively; Mann–Whitney test). mMDSC were more stable towards freezing/thawing, however the difference reached statistical significance (p = 0.039; Mann–Whitney test)

    Journal: Journal of Translational Medicine

    Article Title: Kinetics of human myeloid-derived suppressor cells after blood draw

    doi: 10.1186/s12967-015-0755-y

    Figure Lengend Snippet: Kinetics of gMDSC and mMDSC levels. Frequency of gMDSC ( a ) and mMDSC ( b ) within PBMC after 2, 4, 6 h, overnight rest (ON) and freezing/thawing procedure (defrost). PBMC were stored in RPMI media after the ficoll separation. gMDSC were analysed in 24 patients with chronic viral infections and mMDSC in 17 patients with solid tumors and three HIV infected patients. There was no statistical significant difference in gMDSC frequencies over time (2, 4, 6 h, ON). Statistical significant reduction of gMDSC frequencies after freezing/thawing procedure (p = 0.0001; Mann–Whitney test). The difference in frequencies in mMDSC is not significant after 4 h whereas their numbers were significantly reduced after a 6 h and overnight rest (p = 0.0005 and p = 0.005, respectively; Mann–Whitney test). mMDSC were more stable towards freezing/thawing, however the difference reached statistical significance (p = 0.039; Mann–Whitney test)

    Article Snippet: PBMC isolation, freezing and thawing PBMC were isolated from freshly obtained EDTA blood by Ficoll density gradient centrifugation (Biocoll Separation Solution, Biochrom, Germany).

    Techniques: Infection, MANN-WHITNEY

    (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by Percoll gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.

    Journal: Journal of Virology

    Article Title: Discordance between Bovine Leukemia Virus Tax Immortalization In Vitro and Oncogenicity In Vivo

    doi:

    Figure Lengend Snippet: (A) Phenotype of B cells in BLV-infected sheep. A series of 12 sheep were analyzed to determine and compare the phenotypes of the B-lymphocyte populations within the bloodstream of animals 104, 296, and 480 infected with pBLVTax106+293 (Tax mutant). Three sheep (113, 115, and 116) that were seronegative for BLV were used as controls, whereas six others were infected with viruses exhibiting wild-type behavior during pathogenesis (8, 292, 293, 247, 175, and 235). The different samples were classified in the figure on basis of the proviral loads as determined by semiquantitative PCR. In some lanes (∗), the lysates were diluted 10-fold prior to PCR. PBMCs were isolated form the bloodstream and purified by Percoll gradient centrifugation. The cells were then labeled with monoclonal antibodies 1H4, CC17, and CC125, which recognize surface IgM, CD5, and CD11b, respectively. A similar protocol was applied for labeling the major capsid protein p24 with 4′G9 except that the cells were first cultivated for 24 h to trigger viral expression. Discrimination of the different cell populations was performed by two-color flow cytometry. The data, represented as percentage of the total PBMC population (± the corresponding standard deviation), were deduced from three independent experiments performed over a period of several weeks. When the standard deviation is not indicated (a), the results are the mean values of only two analyses. (B) Titration of the major capsid protein p24 after short-term culture. PBMCs were isolated from the sheep indicated and cultivated for 24 h. Then, the p24 antigen was titrated in the cell culture supernatants by using the ELISA procedure. The data, represented as optical densities, derive from three independent experiments. (C) Expression of CD8 marker on B lymphocytes from sheep 480. PBMCs from six representative sheep infected either with wild-type viruses (292, 175, and 235) or the Tax mutant (104, 296, and 480) were double-labeled with monoclonal antibodies 1H4 and CC63, specific for surface IgM B lymphocytes and CD8, respectively. The cells were then analyzed by two-color flow cytometry, and results from a representative experiment (out of three) are shown as dot plots.

    Article Snippet: Briefly, blood samples were collected by jugular venipuncture, and PBMCs were purified by Percoll density gradient centrifugation (Amersham-Pharmacia).

    Techniques: Infection, Mutagenesis, Polymerase Chain Reaction, Isolation, Purification, Gradient Centrifugation, Labeling, Expressing, Flow Cytometry, Cytometry, Standard Deviation, Titration, Cell Culture, Enzyme-linked Immunosorbent Assay, Marker