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CAR signalling is sufficient for CAR-M mediated-cytokine/chemokine production. A . Experimental scheme of the mixed-species bulk RNAseq set-up. Murine purified CAR-M were incubated for 1 h with the CD19-complex, or with <t>human</t> <t>B</t> cell tumors <t>(Daudi)</t> transduced to express murine CD19 and the phagocytosis probe. Control CAR-M did not receive any treatment. Phagocytic CAR-M were identified based on their YFP low fluorescence and flow cytometry sorted. All three conditions were sequenced together. Analysis was performed by first mapping the reads onto the human genome and excluding all reads mapping there, then performing the mapping on the murine genome. The experiment included 3-5 biological replicates and was performed independently twice. B. Scatterplot representing the mean expression of each gene in the CAR phagocytosing condition (x-axis) compared to the CAR crosslinking condition (y-axis). C . Heatmap representing the relative expression of genes associated to pro-inflammatory pathways that are significantly differentially regulated in phagocytic or crosslinked CAR-M (n = 3-5 independent experiments). D. Absolute concentration of CXCL1, CXCL10, CCL5 and TNFα measured in co-culture of control macrophages (M Empty ) or CAR-M with Eµ-Myc tumor cells for 24 h. CAR-M were also cultured in the presence of the CD19 complex for 24 h (CAR crosslinking) (n= 3 independent replicates, statistical analysis was performed by paired one-way ANOVA). E-F . Schematic experimental set-up (E) showing the transwell assay. Macrophages (Control (M UTD or M Empty ) or CAR-M) and tumor cells were plated at the bottom part while naïve CTV-stained OT-I T cells were added with MC38-OVA on the upper part of the well. For the crosslinking condition, CAR-M were left without tumor cells at the bottom and the CD19 complex was added for 24 h. 72 h later T cell proliferation was measured by CTV dilution by flow cytometry and graphed in (F) (n = 4 independent experiments, statistical analysis was performed by paired one-way ANOVA). Error bars represent mean ± SEM. ***P_<_0.001; **P_<_0.01; *P < 0.05. See Methods for additional experimental details.
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CAR signalling is sufficient for CAR-M mediated-cytokine/chemokine production. A . Experimental scheme of the mixed-species bulk RNAseq set-up. Murine purified CAR-M were incubated for 1 h with the CD19-complex, or with <t>human</t> <t>B</t> cell tumors <t>(Daudi)</t> transduced to express murine CD19 and the phagocytosis probe. Control CAR-M did not receive any treatment. Phagocytic CAR-M were identified based on their YFP low fluorescence and flow cytometry sorted. All three conditions were sequenced together. Analysis was performed by first mapping the reads onto the human genome and excluding all reads mapping there, then performing the mapping on the murine genome. The experiment included 3-5 biological replicates and was performed independently twice. B. Scatterplot representing the mean expression of each gene in the CAR phagocytosing condition (x-axis) compared to the CAR crosslinking condition (y-axis). C . Heatmap representing the relative expression of genes associated to pro-inflammatory pathways that are significantly differentially regulated in phagocytic or crosslinked CAR-M (n = 3-5 independent experiments). D. Absolute concentration of CXCL1, CXCL10, CCL5 and TNFα measured in co-culture of control macrophages (M Empty ) or CAR-M with Eµ-Myc tumor cells for 24 h. CAR-M were also cultured in the presence of the CD19 complex for 24 h (CAR crosslinking) (n= 3 independent replicates, statistical analysis was performed by paired one-way ANOVA). E-F . Schematic experimental set-up (E) showing the transwell assay. Macrophages (Control (M UTD or M Empty ) or CAR-M) and tumor cells were plated at the bottom part while naïve CTV-stained OT-I T cells were added with MC38-OVA on the upper part of the well. For the crosslinking condition, CAR-M were left without tumor cells at the bottom and the CD19 complex was added for 24 h. 72 h later T cell proliferation was measured by CTV dilution by flow cytometry and graphed in (F) (n = 4 independent experiments, statistical analysis was performed by paired one-way ANOVA). Error bars represent mean ± SEM. ***P_<_0.001; **P_<_0.01; *P < 0.05. See Methods for additional experimental details.
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CAR signalling is sufficient for CAR-M mediated-cytokine/chemokine production. A . Experimental scheme of the mixed-species bulk RNAseq set-up. Murine purified CAR-M were incubated for 1 h with the CD19-complex, or with <t>human</t> <t>B</t> cell tumors <t>(Daudi)</t> transduced to express murine CD19 and the phagocytosis probe. Control CAR-M did not receive any treatment. Phagocytic CAR-M were identified based on their YFP low fluorescence and flow cytometry sorted. All three conditions were sequenced together. Analysis was performed by first mapping the reads onto the human genome and excluding all reads mapping there, then performing the mapping on the murine genome. The experiment included 3-5 biological replicates and was performed independently twice. B. Scatterplot representing the mean expression of each gene in the CAR phagocytosing condition (x-axis) compared to the CAR crosslinking condition (y-axis). C . Heatmap representing the relative expression of genes associated to pro-inflammatory pathways that are significantly differentially regulated in phagocytic or crosslinked CAR-M (n = 3-5 independent experiments). D. Absolute concentration of CXCL1, CXCL10, CCL5 and TNFα measured in co-culture of control macrophages (M Empty ) or CAR-M with Eµ-Myc tumor cells for 24 h. CAR-M were also cultured in the presence of the CD19 complex for 24 h (CAR crosslinking) (n= 3 independent replicates, statistical analysis was performed by paired one-way ANOVA). E-F . Schematic experimental set-up (E) showing the transwell assay. Macrophages (Control (M UTD or M Empty ) or CAR-M) and tumor cells were plated at the bottom part while naïve CTV-stained OT-I T cells were added with MC38-OVA on the upper part of the well. For the crosslinking condition, CAR-M were left without tumor cells at the bottom and the CD19 complex was added for 24 h. 72 h later T cell proliferation was measured by CTV dilution by flow cytometry and graphed in (F) (n = 4 independent experiments, statistical analysis was performed by paired one-way ANOVA). Error bars represent mean ± SEM. ***P_<_0.001; **P_<_0.01; *P < 0.05. See Methods for additional experimental details.
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CAR signalling is sufficient for CAR-M mediated-cytokine/chemokine production. A . Experimental scheme of the mixed-species bulk RNAseq set-up. Murine purified CAR-M were incubated for 1 h with the CD19-complex, or with <t>human</t> <t>B</t> cell tumors <t>(Daudi)</t> transduced to express murine CD19 and the phagocytosis probe. Control CAR-M did not receive any treatment. Phagocytic CAR-M were identified based on their YFP low fluorescence and flow cytometry sorted. All three conditions were sequenced together. Analysis was performed by first mapping the reads onto the human genome and excluding all reads mapping there, then performing the mapping on the murine genome. The experiment included 3-5 biological replicates and was performed independently twice. B. Scatterplot representing the mean expression of each gene in the CAR phagocytosing condition (x-axis) compared to the CAR crosslinking condition (y-axis). C . Heatmap representing the relative expression of genes associated to pro-inflammatory pathways that are significantly differentially regulated in phagocytic or crosslinked CAR-M (n = 3-5 independent experiments). D. Absolute concentration of CXCL1, CXCL10, CCL5 and TNFα measured in co-culture of control macrophages (M Empty ) or CAR-M with Eµ-Myc tumor cells for 24 h. CAR-M were also cultured in the presence of the CD19 complex for 24 h (CAR crosslinking) (n= 3 independent replicates, statistical analysis was performed by paired one-way ANOVA). E-F . Schematic experimental set-up (E) showing the transwell assay. Macrophages (Control (M UTD or M Empty ) or CAR-M) and tumor cells were plated at the bottom part while naïve CTV-stained OT-I T cells were added with MC38-OVA on the upper part of the well. For the crosslinking condition, CAR-M were left without tumor cells at the bottom and the CD19 complex was added for 24 h. 72 h later T cell proliferation was measured by CTV dilution by flow cytometry and graphed in (F) (n = 4 independent experiments, statistical analysis was performed by paired one-way ANOVA). Error bars represent mean ± SEM. ***P_<_0.001; **P_<_0.01; *P < 0.05. See Methods for additional experimental details.
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CAR signalling is sufficient for CAR-M mediated-cytokine/chemokine production. A . Experimental scheme of the mixed-species bulk RNAseq set-up. Murine purified CAR-M were incubated for 1 h with the CD19-complex, or with <t>human</t> <t>B</t> cell tumors <t>(Daudi)</t> transduced to express murine CD19 and the phagocytosis probe. Control CAR-M did not receive any treatment. Phagocytic CAR-M were identified based on their YFP low fluorescence and flow cytometry sorted. All three conditions were sequenced together. Analysis was performed by first mapping the reads onto the human genome and excluding all reads mapping there, then performing the mapping on the murine genome. The experiment included 3-5 biological replicates and was performed independently twice. B. Scatterplot representing the mean expression of each gene in the CAR phagocytosing condition (x-axis) compared to the CAR crosslinking condition (y-axis). C . Heatmap representing the relative expression of genes associated to pro-inflammatory pathways that are significantly differentially regulated in phagocytic or crosslinked CAR-M (n = 3-5 independent experiments). D. Absolute concentration of CXCL1, CXCL10, CCL5 and TNFα measured in co-culture of control macrophages (M Empty ) or CAR-M with Eµ-Myc tumor cells for 24 h. CAR-M were also cultured in the presence of the CD19 complex for 24 h (CAR crosslinking) (n= 3 independent replicates, statistical analysis was performed by paired one-way ANOVA). E-F . Schematic experimental set-up (E) showing the transwell assay. Macrophages (Control (M UTD or M Empty ) or CAR-M) and tumor cells were plated at the bottom part while naïve CTV-stained OT-I T cells were added with MC38-OVA on the upper part of the well. For the crosslinking condition, CAR-M were left without tumor cells at the bottom and the CD19 complex was added for 24 h. 72 h later T cell proliferation was measured by CTV dilution by flow cytometry and graphed in (F) (n = 4 independent experiments, statistical analysis was performed by paired one-way ANOVA). Error bars represent mean ± SEM. ***P_<_0.001; **P_<_0.01; *P < 0.05. See Methods for additional experimental details.
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CAR signalling is sufficient for CAR-M mediated-cytokine/chemokine production. A . Experimental scheme of the mixed-species bulk RNAseq set-up. Murine purified CAR-M were incubated for 1 h with the CD19-complex, or with human B cell tumors (Daudi) transduced to express murine CD19 and the phagocytosis probe. Control CAR-M did not receive any treatment. Phagocytic CAR-M were identified based on their YFP low fluorescence and flow cytometry sorted. All three conditions were sequenced together. Analysis was performed by first mapping the reads onto the human genome and excluding all reads mapping there, then performing the mapping on the murine genome. The experiment included 3-5 biological replicates and was performed independently twice. B. Scatterplot representing the mean expression of each gene in the CAR phagocytosing condition (x-axis) compared to the CAR crosslinking condition (y-axis). C . Heatmap representing the relative expression of genes associated to pro-inflammatory pathways that are significantly differentially regulated in phagocytic or crosslinked CAR-M (n = 3-5 independent experiments). D. Absolute concentration of CXCL1, CXCL10, CCL5 and TNFα measured in co-culture of control macrophages (M Empty ) or CAR-M with Eµ-Myc tumor cells for 24 h. CAR-M were also cultured in the presence of the CD19 complex for 24 h (CAR crosslinking) (n= 3 independent replicates, statistical analysis was performed by paired one-way ANOVA). E-F . Schematic experimental set-up (E) showing the transwell assay. Macrophages (Control (M UTD or M Empty ) or CAR-M) and tumor cells were plated at the bottom part while naïve CTV-stained OT-I T cells were added with MC38-OVA on the upper part of the well. For the crosslinking condition, CAR-M were left without tumor cells at the bottom and the CD19 complex was added for 24 h. 72 h later T cell proliferation was measured by CTV dilution by flow cytometry and graphed in (F) (n = 4 independent experiments, statistical analysis was performed by paired one-way ANOVA). Error bars represent mean ± SEM. ***P_<_0.001; **P_<_0.01; *P < 0.05. See Methods for additional experimental details.

Journal: bioRxiv

Article Title: CAR-MACROPHAGES ACTIVATE ANTI TUMOR T CELLS IN THE ABSENCE OF PHAGOCYTOSIS

doi: 10.64898/2026.03.11.710792

Figure Lengend Snippet: CAR signalling is sufficient for CAR-M mediated-cytokine/chemokine production. A . Experimental scheme of the mixed-species bulk RNAseq set-up. Murine purified CAR-M were incubated for 1 h with the CD19-complex, or with human B cell tumors (Daudi) transduced to express murine CD19 and the phagocytosis probe. Control CAR-M did not receive any treatment. Phagocytic CAR-M were identified based on their YFP low fluorescence and flow cytometry sorted. All three conditions were sequenced together. Analysis was performed by first mapping the reads onto the human genome and excluding all reads mapping there, then performing the mapping on the murine genome. The experiment included 3-5 biological replicates and was performed independently twice. B. Scatterplot representing the mean expression of each gene in the CAR phagocytosing condition (x-axis) compared to the CAR crosslinking condition (y-axis). C . Heatmap representing the relative expression of genes associated to pro-inflammatory pathways that are significantly differentially regulated in phagocytic or crosslinked CAR-M (n = 3-5 independent experiments). D. Absolute concentration of CXCL1, CXCL10, CCL5 and TNFα measured in co-culture of control macrophages (M Empty ) or CAR-M with Eµ-Myc tumor cells for 24 h. CAR-M were also cultured in the presence of the CD19 complex for 24 h (CAR crosslinking) (n= 3 independent replicates, statistical analysis was performed by paired one-way ANOVA). E-F . Schematic experimental set-up (E) showing the transwell assay. Macrophages (Control (M UTD or M Empty ) or CAR-M) and tumor cells were plated at the bottom part while naïve CTV-stained OT-I T cells were added with MC38-OVA on the upper part of the well. For the crosslinking condition, CAR-M were left without tumor cells at the bottom and the CD19 complex was added for 24 h. 72 h later T cell proliferation was measured by CTV dilution by flow cytometry and graphed in (F) (n = 4 independent experiments, statistical analysis was performed by paired one-way ANOVA). Error bars represent mean ± SEM. ***P_<_0.001; **P_<_0.01; *P < 0.05. See Methods for additional experimental details.

Article Snippet: Human B cell Daudi tumors (ATCC, CCL-213) were retrovirally transduced to express the DEVD phagocytosis reporter as well as murine CD19 and CD20 molecules.

Techniques: RNA sequencing, Purification, Incubation, Control, Fluorescence, Flow Cytometry, Expressing, Concentration Assay, Co-Culture Assay, Cell Culture, Transwell Assay, Staining