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(A) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA and the GEP of spleen CD19 low CD138 + Plasma-cells from C57BL6 mice and C-IM cancer cells. (B) Pathway analysis enrichment (Metacore) of the GEP of C-IM cancer cells compared to spleen CD19 low CD138 + Plasma-cells. The top 5 enriched pathways are shown. (C) RNAseq expression data for genes related to <t>the</t> <t>IL-6</t> signaling pathway, Socs3 and Il6st . GCB: Germinal Center B-cells, B1: B1 B-cells, FOB: Follicular B-cells, MZB: Marginal Zone B-cells, SPLPC: Spleen Plasma-cells, BMPC: Bone Marrow Plasma-cells, SPLPB: Spleen Plasmablasts , C-IM : GFP + hCD2 + C-IM cancer cells (FACS purified). (D) Representative flow cytometric analysis of the proliferation profiles illustrating the frequency of (pre)plasmablasts (CD138 + ) on day 2, 3, and 4 of LPS stimulation of B-cells from CD19cre IKK2ca stopFL MYC stopFL (IKK2caMYC) in vitro , in the presence of either control antibody (control Ig) or ant-IL6 neutralizing (anti-IL6). Cell Trace Violet (CTV) dye dilution was used to assess proliferation. (E) MFI of pSTAT3 in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes. Black circles represent control mice (WT), yellow circles CD19cre IKK2ca stopFL mice (IKK2), blue circles CD19cre MYC stopFL (MYC), and red circles CD19cre IKK2ca stopFL MYC stopFL (IKK2MYC). White background: control Ig, dashed background: anti-IL6. (F) Fold change (anti-IL6/control Ig) of the fraction of cleaved caspase 3 + cells within B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). (G) MFI of BCL-xL in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). White background: control Ig, dashed background: anti-IL6. (H) MFI of MCL-1 in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). White background: control Ig, dashed background: anti-IL6. (I) Principal component (PC) analysis plots of RNA-seq analysis of activated B cells (B220 + ) and B220 low CD138 + (pre)plasmablasts at day 3 of in vitro culture of B-cells from CD19cre IKK2ca stopFL MYC stopFL (IKK2.MYC). (J) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA and the GEP of B220 low CD138 + (pre)plasmablasts at day 3 of in vitro culture of B-cells from CD19cre IKK2ca stopFL MYC stopFL in the presence of control Ig (IKK2.MYC control Ig) or ant-IL6 antibody (IKK2.MYC anti IL6). (K) Cancer free survival curve for C-IM mice treated with control Ig (solid red line) or ant-IL6 antibody (dashed red line).
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(A) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA and the GEP of spleen CD19 low CD138 + Plasma-cells from C57BL6 mice and C-IM cancer cells. (B) Pathway analysis enrichment (Metacore) of the GEP of C-IM cancer cells compared to spleen CD19 low CD138 + Plasma-cells. The top 5 enriched pathways are shown. (C) RNAseq expression data for genes related to the IL-6 signaling pathway, Socs3 and Il6st . GCB: Germinal Center B-cells, B1: B1 B-cells, FOB: Follicular B-cells, MZB: Marginal Zone B-cells, SPLPC: Spleen Plasma-cells, BMPC: Bone Marrow Plasma-cells, SPLPB: Spleen Plasmablasts , C-IM : GFP + hCD2 + C-IM cancer cells (FACS purified). (D) Representative flow cytometric analysis of the proliferation profiles illustrating the frequency of (pre)plasmablasts (CD138 + ) on day 2, 3, and 4 of LPS stimulation of B-cells from CD19cre IKK2ca stopFL MYC stopFL (IKK2caMYC) in vitro , in the presence of either control antibody (control Ig) or ant-IL6 neutralizing (anti-IL6). Cell Trace Violet (CTV) dye dilution was used to assess proliferation. (E) MFI of pSTAT3 in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes. Black circles represent control mice (WT), yellow circles CD19cre IKK2ca stopFL mice (IKK2), blue circles CD19cre MYC stopFL (MYC), and red circles CD19cre IKK2ca stopFL MYC stopFL (IKK2MYC). White background: control Ig, dashed background: anti-IL6. (F) Fold change (anti-IL6/control Ig) of the fraction of cleaved caspase 3 + cells within B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). (G) MFI of BCL-xL in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). White background: control Ig, dashed background: anti-IL6. (H) MFI of MCL-1 in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). White background: control Ig, dashed background: anti-IL6. (I) Principal component (PC) analysis plots of RNA-seq analysis of activated B cells (B220 + ) and B220 low CD138 + (pre)plasmablasts at day 3 of in vitro culture of B-cells from CD19cre IKK2ca stopFL MYC stopFL (IKK2.MYC). (J) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA and the GEP of B220 low CD138 + (pre)plasmablasts at day 3 of in vitro culture of B-cells from CD19cre IKK2ca stopFL MYC stopFL in the presence of control Ig (IKK2.MYC control Ig) or ant-IL6 antibody (IKK2.MYC anti IL6). (K) Cancer free survival curve for C-IM mice treated with control Ig (solid red line) or ant-IL6 antibody (dashed red line).

Journal: bioRxiv

Article Title: Co-activation of NF-κB and MYC renders cancer cells addicted to IL6 for survival and phenotypic stability

doi: 10.1101/2020.04.12.038414

Figure Lengend Snippet: (A) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA and the GEP of spleen CD19 low CD138 + Plasma-cells from C57BL6 mice and C-IM cancer cells. (B) Pathway analysis enrichment (Metacore) of the GEP of C-IM cancer cells compared to spleen CD19 low CD138 + Plasma-cells. The top 5 enriched pathways are shown. (C) RNAseq expression data for genes related to the IL-6 signaling pathway, Socs3 and Il6st . GCB: Germinal Center B-cells, B1: B1 B-cells, FOB: Follicular B-cells, MZB: Marginal Zone B-cells, SPLPC: Spleen Plasma-cells, BMPC: Bone Marrow Plasma-cells, SPLPB: Spleen Plasmablasts , C-IM : GFP + hCD2 + C-IM cancer cells (FACS purified). (D) Representative flow cytometric analysis of the proliferation profiles illustrating the frequency of (pre)plasmablasts (CD138 + ) on day 2, 3, and 4 of LPS stimulation of B-cells from CD19cre IKK2ca stopFL MYC stopFL (IKK2caMYC) in vitro , in the presence of either control antibody (control Ig) or ant-IL6 neutralizing (anti-IL6). Cell Trace Violet (CTV) dye dilution was used to assess proliferation. (E) MFI of pSTAT3 in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes. Black circles represent control mice (WT), yellow circles CD19cre IKK2ca stopFL mice (IKK2), blue circles CD19cre MYC stopFL (MYC), and red circles CD19cre IKK2ca stopFL MYC stopFL (IKK2MYC). White background: control Ig, dashed background: anti-IL6. (F) Fold change (anti-IL6/control Ig) of the fraction of cleaved caspase 3 + cells within B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). (G) MFI of BCL-xL in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). White background: control Ig, dashed background: anti-IL6. (H) MFI of MCL-1 in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). White background: control Ig, dashed background: anti-IL6. (I) Principal component (PC) analysis plots of RNA-seq analysis of activated B cells (B220 + ) and B220 low CD138 + (pre)plasmablasts at day 3 of in vitro culture of B-cells from CD19cre IKK2ca stopFL MYC stopFL (IKK2.MYC). (J) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA and the GEP of B220 low CD138 + (pre)plasmablasts at day 3 of in vitro culture of B-cells from CD19cre IKK2ca stopFL MYC stopFL in the presence of control Ig (IKK2.MYC control Ig) or ant-IL6 antibody (IKK2.MYC anti IL6). (K) Cancer free survival curve for C-IM mice treated with control Ig (solid red line) or ant-IL6 antibody (dashed red line).

Article Snippet: To study the impact of IL6 on cancer progression, C-IM mice were treated with InVivoMAb anti-mouse IL-6 (bioXcell), or a control antibody (InVivoMAb rat IgG1 isotype control, anti-horseradish peroxidase; bioXcell).

Techniques: Expressing, Purification, In Vitro, RNA Sequencing Assay