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( a ) <t>PI3Kγ</t> and STING expression at lymph node and pancreatic tumor from KPC mice compared to C57BL/6 mice. ( b ) PI3Kγ and STING expression at B cell, bone marrow derived dendritic cells and bone marrow derived macrophages from KPC mice and C57BL/6 mice. ( c ) Phosphorylation of IRF3 at splenic B cells, bone marrow derived dendritic cells (BMDCs), bone marrow derived macrophages (BMDMs) and CD4 T cells derived from KPC mice and THP-1 hSTING HAQ cells after treatments with or without MSA-2 or IPI-549. ( d ) Quantification and representative flow cytometry analysis of IL35 + and IL10 + Breg cells (from KPC spleen) after treatments with or without MSA-2 or IPI-549. ( e ) Quantification of mouse Interferon-β concentration of bone marrow derived dendritic cells (BMDCs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( f, g ) Quantification of human Interferon-β concentration from THP-1 R232 hSTING R232 after treatments with or without MSA-2 or IPI-549. ( g ) Quantification of STING activation by reporter assay from THP1-Blue TM ISG by measuring optical density at 655 nm after treatments with or without MSA-2 or IPI-549. ( h ) Quantification of human Interferon-β concentration from THP-1 hSTING KO after treatments with or without MSA-2 or IPI-549. ( i, j ) Quantification of CD86 geometric mean fluorescent intensity of bone marrow derived dendritic cells (BMDCs) bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( k ) Quantification of RAW264.7 M1 polarization ratios after treatments with or without MSA-2 or IPI-549. ( l ) Quantification of TNF-α concentration from RAW264.7 after treatments with or without MSA-2 or IPI-549. Data for quantification are shown as mean ± SD, n = 3. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s pairwise comparisons or by Student’s unpaired T-test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation; n.s., no statistical significance. For ( d ) statistical comparisons are conducted between MSA-2 group with other groups. For ( e-l ), statistical comparisons are conducted between MSA-2 and MSA-2+IPI-549 groups with control group.
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( a ) <t>PI3Kγ</t> and STING expression at lymph node and pancreatic tumor from KPC mice compared to C57BL/6 mice. ( b ) PI3Kγ and STING expression at B cell, bone marrow derived dendritic cells and bone marrow derived macrophages from KPC mice and C57BL/6 mice. ( c ) Phosphorylation of IRF3 at splenic B cells, bone marrow derived dendritic cells (BMDCs), bone marrow derived macrophages (BMDMs) and CD4 T cells derived from KPC mice and THP-1 hSTING HAQ cells after treatments with or without MSA-2 or IPI-549. ( d ) Quantification and representative flow cytometry analysis of IL35 + and IL10 + Breg cells (from KPC spleen) after treatments with or without MSA-2 or IPI-549. ( e ) Quantification of mouse Interferon-β concentration of bone marrow derived dendritic cells (BMDCs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( f, g ) Quantification of human Interferon-β concentration from THP-1 R232 hSTING R232 after treatments with or without MSA-2 or IPI-549. ( g ) Quantification of STING activation by reporter assay from THP1-Blue TM ISG by measuring optical density at 655 nm after treatments with or without MSA-2 or IPI-549. ( h ) Quantification of human Interferon-β concentration from THP-1 hSTING KO after treatments with or without MSA-2 or IPI-549. ( i, j ) Quantification of CD86 geometric mean fluorescent intensity of bone marrow derived dendritic cells (BMDCs) bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( k ) Quantification of RAW264.7 M1 polarization ratios after treatments with or without MSA-2 or IPI-549. ( l ) Quantification of TNF-α concentration from RAW264.7 after treatments with or without MSA-2 or IPI-549. Data for quantification are shown as mean ± SD, n = 3. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s pairwise comparisons or by Student’s unpaired T-test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation; n.s., no statistical significance. For ( d ) statistical comparisons are conducted between MSA-2 group with other groups. For ( e-l ), statistical comparisons are conducted between MSA-2 and MSA-2+IPI-549 groups with control group.
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( a ) <t>PI3Kγ</t> and STING expression at lymph node and pancreatic tumor from KPC mice compared to C57BL/6 mice. ( b ) PI3Kγ and STING expression at B cell, bone marrow derived dendritic cells and bone marrow derived macrophages from KPC mice and C57BL/6 mice. ( c ) Phosphorylation of IRF3 at splenic B cells, bone marrow derived dendritic cells (BMDCs), bone marrow derived macrophages (BMDMs) and CD4 T cells derived from KPC mice and THP-1 hSTING HAQ cells after treatments with or without MSA-2 or IPI-549. ( d ) Quantification and representative flow cytometry analysis of IL35 + and IL10 + Breg cells (from KPC spleen) after treatments with or without MSA-2 or IPI-549. ( e ) Quantification of mouse Interferon-β concentration of bone marrow derived dendritic cells (BMDCs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( f, g ) Quantification of human Interferon-β concentration from THP-1 R232 hSTING R232 after treatments with or without MSA-2 or IPI-549. ( g ) Quantification of STING activation by reporter assay from THP1-Blue TM ISG by measuring optical density at 655 nm after treatments with or without MSA-2 or IPI-549. ( h ) Quantification of human Interferon-β concentration from THP-1 hSTING KO after treatments with or without MSA-2 or IPI-549. ( i, j ) Quantification of CD86 geometric mean fluorescent intensity of bone marrow derived dendritic cells (BMDCs) bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( k ) Quantification of RAW264.7 M1 polarization ratios after treatments with or without MSA-2 or IPI-549. ( l ) Quantification of TNF-α concentration from RAW264.7 after treatments with or without MSA-2 or IPI-549. Data for quantification are shown as mean ± SD, n = 3. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s pairwise comparisons or by Student’s unpaired T-test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation; n.s., no statistical significance. For ( d ) statistical comparisons are conducted between MSA-2 group with other groups. For ( e-l ), statistical comparisons are conducted between MSA-2 and MSA-2+IPI-549 groups with control group.
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( a ) <t>PI3Kγ</t> and STING expression at lymph node and pancreatic tumor from KPC mice compared to C57BL/6 mice. ( b ) PI3Kγ and STING expression at B cell, bone marrow derived dendritic cells and bone marrow derived macrophages from KPC mice and C57BL/6 mice. ( c ) Phosphorylation of IRF3 at splenic B cells, bone marrow derived dendritic cells (BMDCs), bone marrow derived macrophages (BMDMs) and CD4 T cells derived from KPC mice and THP-1 hSTING HAQ cells after treatments with or without MSA-2 or IPI-549. ( d ) Quantification and representative flow cytometry analysis of IL35 + and IL10 + Breg cells (from KPC spleen) after treatments with or without MSA-2 or IPI-549. ( e ) Quantification of mouse Interferon-β concentration of bone marrow derived dendritic cells (BMDCs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( f, g ) Quantification of human Interferon-β concentration from THP-1 R232 hSTING R232 after treatments with or without MSA-2 or IPI-549. ( g ) Quantification of STING activation by reporter assay from THP1-Blue TM ISG by measuring optical density at 655 nm after treatments with or without MSA-2 or IPI-549. ( h ) Quantification of human Interferon-β concentration from THP-1 hSTING KO after treatments with or without MSA-2 or IPI-549. ( i, j ) Quantification of CD86 geometric mean fluorescent intensity of bone marrow derived dendritic cells (BMDCs) bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( k ) Quantification of RAW264.7 M1 polarization ratios after treatments with or without MSA-2 or IPI-549. ( l ) Quantification of TNF-α concentration from RAW264.7 after treatments with or without MSA-2 or IPI-549. Data for quantification are shown as mean ± SD, n = 3. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s pairwise comparisons or by Student’s unpaired T-test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation; n.s., no statistical significance. For ( d ) statistical comparisons are conducted between MSA-2 group with other groups. For ( e-l ), statistical comparisons are conducted between MSA-2 and MSA-2+IPI-549 groups with control group.
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( a ) <t>PI3Kγ</t> and STING expression at lymph node and pancreatic tumor from KPC mice compared to C57BL/6 mice. ( b ) PI3Kγ and STING expression at B cell, bone marrow derived dendritic cells and bone marrow derived macrophages from KPC mice and C57BL/6 mice. ( c ) Phosphorylation of IRF3 at splenic B cells, bone marrow derived dendritic cells (BMDCs), bone marrow derived macrophages (BMDMs) and CD4 T cells derived from KPC mice and THP-1 hSTING HAQ cells after treatments with or without MSA-2 or IPI-549. ( d ) Quantification and representative flow cytometry analysis of IL35 + and IL10 + Breg cells (from KPC spleen) after treatments with or without MSA-2 or IPI-549. ( e ) Quantification of mouse Interferon-β concentration of bone marrow derived dendritic cells (BMDCs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( f, g ) Quantification of human Interferon-β concentration from THP-1 R232 hSTING R232 after treatments with or without MSA-2 or IPI-549. ( g ) Quantification of STING activation by reporter assay from THP1-Blue TM ISG by measuring optical density at 655 nm after treatments with or without MSA-2 or IPI-549. ( h ) Quantification of human Interferon-β concentration from THP-1 hSTING KO after treatments with or without MSA-2 or IPI-549. ( i, j ) Quantification of CD86 geometric mean fluorescent intensity of bone marrow derived dendritic cells (BMDCs) bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( k ) Quantification of RAW264.7 M1 polarization ratios after treatments with or without MSA-2 or IPI-549. ( l ) Quantification of TNF-α concentration from RAW264.7 after treatments with or without MSA-2 or IPI-549. Data for quantification are shown as mean ± SD, n = 3. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s pairwise comparisons or by Student’s unpaired T-test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation; n.s., no statistical significance. For ( d ) statistical comparisons are conducted between MSA-2 group with other groups. For ( e-l ), statistical comparisons are conducted between MSA-2 and MSA-2+IPI-549 groups with control group.
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( a ) <t>PI3Kγ</t> and STING expression at lymph node and pancreatic tumor from KPC mice compared to C57BL/6 mice. ( b ) PI3Kγ and STING expression at B cell, bone marrow derived dendritic cells and bone marrow derived macrophages from KPC mice and C57BL/6 mice. ( c ) Phosphorylation of IRF3 at splenic B cells, bone marrow derived dendritic cells (BMDCs), bone marrow derived macrophages (BMDMs) and CD4 T cells derived from KPC mice and THP-1 hSTING HAQ cells after treatments with or without MSA-2 or IPI-549. ( d ) Quantification and representative flow cytometry analysis of IL35 + and IL10 + Breg cells (from KPC spleen) after treatments with or without MSA-2 or IPI-549. ( e ) Quantification of mouse Interferon-β concentration of bone marrow derived dendritic cells (BMDCs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( f, g ) Quantification of human Interferon-β concentration from THP-1 R232 hSTING R232 after treatments with or without MSA-2 or IPI-549. ( g ) Quantification of STING activation by reporter assay from THP1-Blue TM ISG by measuring optical density at 655 nm after treatments with or without MSA-2 or IPI-549. ( h ) Quantification of human Interferon-β concentration from THP-1 hSTING KO after treatments with or without MSA-2 or IPI-549. ( i, j ) Quantification of CD86 geometric mean fluorescent intensity of bone marrow derived dendritic cells (BMDCs) bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( k ) Quantification of RAW264.7 M1 polarization ratios after treatments with or without MSA-2 or IPI-549. ( l ) Quantification of TNF-α concentration from RAW264.7 after treatments with or without MSA-2 or IPI-549. Data for quantification are shown as mean ± SD, n = 3. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s pairwise comparisons or by Student’s unpaired T-test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation; n.s., no statistical significance. For ( d ) statistical comparisons are conducted between MSA-2 group with other groups. For ( e-l ), statistical comparisons are conducted between MSA-2 and MSA-2+IPI-549 groups with control group.
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( a ) PI3Kγ and STING expression at lymph node and pancreatic tumor from KPC mice compared to C57BL/6 mice. ( b ) PI3Kγ and STING expression at B cell, bone marrow derived dendritic cells and bone marrow derived macrophages from KPC mice and C57BL/6 mice. ( c ) Phosphorylation of IRF3 at splenic B cells, bone marrow derived dendritic cells (BMDCs), bone marrow derived macrophages (BMDMs) and CD4 T cells derived from KPC mice and THP-1 hSTING HAQ cells after treatments with or without MSA-2 or IPI-549. ( d ) Quantification and representative flow cytometry analysis of IL35 + and IL10 + Breg cells (from KPC spleen) after treatments with or without MSA-2 or IPI-549. ( e ) Quantification of mouse Interferon-β concentration of bone marrow derived dendritic cells (BMDCs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( f, g ) Quantification of human Interferon-β concentration from THP-1 R232 hSTING R232 after treatments with or without MSA-2 or IPI-549. ( g ) Quantification of STING activation by reporter assay from THP1-Blue TM ISG by measuring optical density at 655 nm after treatments with or without MSA-2 or IPI-549. ( h ) Quantification of human Interferon-β concentration from THP-1 hSTING KO after treatments with or without MSA-2 or IPI-549. ( i, j ) Quantification of CD86 geometric mean fluorescent intensity of bone marrow derived dendritic cells (BMDCs) bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( k ) Quantification of RAW264.7 M1 polarization ratios after treatments with or without MSA-2 or IPI-549. ( l ) Quantification of TNF-α concentration from RAW264.7 after treatments with or without MSA-2 or IPI-549. Data for quantification are shown as mean ± SD, n = 3. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s pairwise comparisons or by Student’s unpaired T-test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation; n.s., no statistical significance. For ( d ) statistical comparisons are conducted between MSA-2 group with other groups. For ( e-l ), statistical comparisons are conducted between MSA-2 and MSA-2+IPI-549 groups with control group.

Journal: bioRxiv

Article Title: Dual Targeting of STING and PI3Kγ Eliminates Regulatory B Cells to Overcome STING Resistance for Pancreatic Cancer Immunotherapy

doi: 10.1101/2024.02.14.580378

Figure Lengend Snippet: ( a ) PI3Kγ and STING expression at lymph node and pancreatic tumor from KPC mice compared to C57BL/6 mice. ( b ) PI3Kγ and STING expression at B cell, bone marrow derived dendritic cells and bone marrow derived macrophages from KPC mice and C57BL/6 mice. ( c ) Phosphorylation of IRF3 at splenic B cells, bone marrow derived dendritic cells (BMDCs), bone marrow derived macrophages (BMDMs) and CD4 T cells derived from KPC mice and THP-1 hSTING HAQ cells after treatments with or without MSA-2 or IPI-549. ( d ) Quantification and representative flow cytometry analysis of IL35 + and IL10 + Breg cells (from KPC spleen) after treatments with or without MSA-2 or IPI-549. ( e ) Quantification of mouse Interferon-β concentration of bone marrow derived dendritic cells (BMDCs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( f, g ) Quantification of human Interferon-β concentration from THP-1 R232 hSTING R232 after treatments with or without MSA-2 or IPI-549. ( g ) Quantification of STING activation by reporter assay from THP1-Blue TM ISG by measuring optical density at 655 nm after treatments with or without MSA-2 or IPI-549. ( h ) Quantification of human Interferon-β concentration from THP-1 hSTING KO after treatments with or without MSA-2 or IPI-549. ( i, j ) Quantification of CD86 geometric mean fluorescent intensity of bone marrow derived dendritic cells (BMDCs) bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( k ) Quantification of RAW264.7 M1 polarization ratios after treatments with or without MSA-2 or IPI-549. ( l ) Quantification of TNF-α concentration from RAW264.7 after treatments with or without MSA-2 or IPI-549. Data for quantification are shown as mean ± SD, n = 3. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s pairwise comparisons or by Student’s unpaired T-test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation; n.s., no statistical significance. For ( d ) statistical comparisons are conducted between MSA-2 group with other groups. For ( e-l ), statistical comparisons are conducted between MSA-2 and MSA-2+IPI-549 groups with control group.

Article Snippet: PI3Kγ binding affinity was evaluated using PI3Kγ (p110γ/PIK3R5) assay kit from BPS Bioscience, following the provided protocol.

Techniques: Expressing, Derivative Assay, Flow Cytometry, Concentration Assay, Activation Assay, Reporter Assay, Standard Deviation, Control