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Vaccination with TGFβ-derived peptides increases the tumoral-infiltration of CD8 + T cells and polarizes tumor-associated macrophages from an M2-like to an M1-like phenotype. Pan02 tumor-bearing mice (n=8 per group) were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested on day 33, pooled in pairs among treatment groups, and analyzed by flow cytometry. Bar plots show (A) cancer cells gated as CD45 − CD31 − FAP − , (B) leukocytes gated as CD45 + CD31 − , (C) endothelial cells gated as CD45 − CD31 + , (D) CD3 + T cells gated as CD45 + CD3 + , (E) CD8 + T cells gated as CD45 + CD3 + CD8 + CD4 − , (H) CD4 + T cells gated as CD45 + CD3 + CD8 − CD4 + , (I) CD8/CD4 ratio calculated by dividing the percentage of CD8 + T cells among CD3 + cells by the percentage of CD4 + T cells among CD3 + cells, (J) Tregs gated as CD45 + CD3 + CD8 − CD4 + CD25 + FoxP3 + , (K) CD8/Treg ratio calculated by dividing the percentage of CD8 + T cells among CD3 + cells by the percentage of Tregs among CD3 + cells, (L) macrophages gated as <t>CD11b</t> + F4/80 + , (M) M1 macrophages gated as CD11b + F4/80 + mannose receptor (MR) − , (N) M2 macrophages gated as CD11b + F4/80 + MR + and (O) M1/M2 ratio calculated by dividing the percentage of M1 macrophages by the percentage of M2 macrophages in the tumor of untreated or mice treated with the TGFβ vaccine. All populations were gated on single live cells. Gating strategy can be found in . Data are presented as mean±SEM. Dots represent pooled tumors (n=3–4 per group). (F) and (G) Representative dot plots of CD4 + and CD8 + cells in the CD3 + population of (F) untreated or (G) TGFβ vaccine-treated mice. (P) Representative histograms of MR on macrophages in untreated mice or mice treated with the TGFβ vaccine shown in (M) and (N). ns, not significant; *p<0.05 and **p<0.01 according to unpaired two-tailed t test. TGFβ, transforming growth factor-β; Tregs, regulatory T cells.
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Vaccination with TGFβ-derived peptides increases the tumoral-infiltration of CD8 + T cells and polarizes tumor-associated macrophages from an M2-like to an M1-like phenotype. Pan02 tumor-bearing mice (n=8 per group) were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested on day 33, pooled in pairs among treatment groups, and analyzed by flow cytometry. Bar plots show (A) cancer cells gated as CD45 − CD31 − FAP − , (B) leukocytes gated as CD45 + CD31 − , (C) endothelial cells gated as CD45 − CD31 + , (D) CD3 + T cells gated as CD45 + CD3 + , (E) CD8 + T cells gated as CD45 + CD3 + CD8 + CD4 − , (H) CD4 + T cells gated as CD45 + CD3 + CD8 − CD4 + , (I) CD8/CD4 ratio calculated by dividing the percentage of CD8 + T cells among CD3 + cells by the percentage of CD4 + T cells among CD3 + cells, (J) Tregs gated as CD45 + CD3 + CD8 − CD4 + CD25 + FoxP3 + , (K) CD8/Treg ratio calculated by dividing the percentage of CD8 + T cells among CD3 + cells by the percentage of Tregs among CD3 + cells, (L) macrophages gated as CD11b + F4/80 + , (M) M1 macrophages gated as CD11b + F4/80 + mannose receptor (MR) − , (N) M2 macrophages gated as CD11b + F4/80 + MR + and (O) M1/M2 ratio calculated by dividing the percentage of M1 macrophages by the percentage of M2 macrophages in the tumor of untreated or mice treated with the TGFβ vaccine. All populations were gated on single live cells. Gating strategy can be found in . Data are presented as mean±SEM. Dots represent pooled tumors (n=3–4 per group). (F) and (G) Representative dot plots of CD4 + and CD8 + cells in the CD3 + population of (F) untreated or (G) TGFβ vaccine-treated mice. (P) Representative histograms of MR on macrophages in untreated mice or mice treated with the TGFβ vaccine shown in (M) and (N). ns, not significant; *p<0.05 and **p<0.01 according to unpaired two-tailed t test. TGFβ, transforming growth factor-β; Tregs, regulatory T cells.

Journal: Journal for Immunotherapy of Cancer

Article Title: TGFβ-derived immune modulatory vaccine: targeting the immunosuppressive and fibrotic tumor microenvironment in a murine model of pancreatic cancer

doi: 10.1136/jitc-2022-005491

Figure Lengend Snippet: Vaccination with TGFβ-derived peptides increases the tumoral-infiltration of CD8 + T cells and polarizes tumor-associated macrophages from an M2-like to an M1-like phenotype. Pan02 tumor-bearing mice (n=8 per group) were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested on day 33, pooled in pairs among treatment groups, and analyzed by flow cytometry. Bar plots show (A) cancer cells gated as CD45 − CD31 − FAP − , (B) leukocytes gated as CD45 + CD31 − , (C) endothelial cells gated as CD45 − CD31 + , (D) CD3 + T cells gated as CD45 + CD3 + , (E) CD8 + T cells gated as CD45 + CD3 + CD8 + CD4 − , (H) CD4 + T cells gated as CD45 + CD3 + CD8 − CD4 + , (I) CD8/CD4 ratio calculated by dividing the percentage of CD8 + T cells among CD3 + cells by the percentage of CD4 + T cells among CD3 + cells, (J) Tregs gated as CD45 + CD3 + CD8 − CD4 + CD25 + FoxP3 + , (K) CD8/Treg ratio calculated by dividing the percentage of CD8 + T cells among CD3 + cells by the percentage of Tregs among CD3 + cells, (L) macrophages gated as CD11b + F4/80 + , (M) M1 macrophages gated as CD11b + F4/80 + mannose receptor (MR) − , (N) M2 macrophages gated as CD11b + F4/80 + MR + and (O) M1/M2 ratio calculated by dividing the percentage of M1 macrophages by the percentage of M2 macrophages in the tumor of untreated or mice treated with the TGFβ vaccine. All populations were gated on single live cells. Gating strategy can be found in . Data are presented as mean±SEM. Dots represent pooled tumors (n=3–4 per group). (F) and (G) Representative dot plots of CD4 + and CD8 + cells in the CD3 + population of (F) untreated or (G) TGFβ vaccine-treated mice. (P) Representative histograms of MR on macrophages in untreated mice or mice treated with the TGFβ vaccine shown in (M) and (N). ns, not significant; *p<0.05 and **p<0.01 according to unpaired two-tailed t test. TGFβ, transforming growth factor-β; Tregs, regulatory T cells.

Article Snippet: The following antibodies/dies (purchased from BioLegend unless otherwise stated) were used for flow cytometry: CD45-PE-Cy7, CD31-FITC, FAP-biotin (R&D system), streptavidin-APC, CD90-BV605, PDPN-APC, Ly6C-AF700, CD26-PerCP-Cy5.5 (eBioscience), αSMA-Cy3 (Sigma), CD11b-PE-Cy7, CD11b-Pacific Blue, F4/80-APC, F4/80-FITC, MR-PE, MR-PE-Cy7, Ly6C-PerCP-Cy5.5, Ly6G-APC-Cy7, Arg1-PE (R&D system), PDL1-APC (BD Biosciences), CD45-FITC, CD3-AF700, CD4-BV421, CD8-BV605 (BD Biosciences), CD25-PE-Cy7, FoxP3-APC and carboxyfluorescein succinimidyl ester (CFSE, Sigma Aldrich).

Techniques: Derivative Assay, Flow Cytometry, Two Tailed Test

TGFβ vaccination results in reduced intratumoral secretion of TGFβ and reduces immunosuppression of T cells and macrophages in the tumor microenvironment. Pan02 tumor-bearing mice were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested (n=4 per group) on day 24 and pooled in pairs among treatment groups. Tumor-conditioned media (TCM) secreted by 0.1×10 6 cells from the tumor digest during a 48 hours culture was harvested. (A) Quantification of TGFβ1 protein levels in TCM from untreated and TGFβ-vaccinated mice. (B) Effect of TCM from untreated or TGFβ-vaccinated mice on the proliferation of anti-CD3/CD28-stimualted naïve splenocytes from an untreated tumor-free mouse. Naïve splenocytes were CFSE-labeled and activated with Dynabeads Mouse T-Activator CD3/CD28 (1:1 ratio) for 48 hours in the presence or absence of TCM generated from tumors from untreated or TGFβ-vaccinated mice. Proliferation of live CD3 + cells was measured by flow cytometry. Gating strategy can be found in . (B, left) Proliferation index in CD3 + cells across culture conditions. (B, right) Representative histograms of CFSE staining in live CD3 + cells (B). (C–E) Effect of TCM from untreated or TGFβ-vaccinated mice on the phenotype of bone-marrow derived macrophages (BMDM) from an untreated tumor-free mouse that were polarized with IL-4 to an M2-like phenotype. BMDM were cultured for 24 hours in the presence of TCM generated from tumors from untreated or TGFβ-vaccinated mice. The phenotype of BMDM was assessed by flow cytometry. (C, top) M1/M2 ratio in BMDM cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. Macrophages were gated as CD11b + F4/80 + , M1 and M2 macrophages were gated as CD11b + F4/80 + mannose receptor (MR) − and CD11b + F4/80 + MR + , respectively. Gating strategy can be found in . (C, bottom) Representative histograms of MR on macrophages across culture conditions shown in (C, top). (D, top) Mean fluorescence intensity (MFI) of Arg1 macrophages cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. (D, bottom) Representative histograms of Arg1 on macrophages across culture conditions shown in (D, top). (E, top) MFI of PD-L1 macrophages cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. (E, bottom) Representative histograms of PD-L1 on macrophages across culture conditions shown in (E, top). Bar plots are presented as mean±SD. Dots represent data derived from TCM generated from pooled tumors (n=2 per group). Arg1, arginase-1; IL, interleukin; PD-L1, programmed death-ligand 1; TGFβ, transforming growth factor-β.

Journal: Journal for Immunotherapy of Cancer

Article Title: TGFβ-derived immune modulatory vaccine: targeting the immunosuppressive and fibrotic tumor microenvironment in a murine model of pancreatic cancer

doi: 10.1136/jitc-2022-005491

Figure Lengend Snippet: TGFβ vaccination results in reduced intratumoral secretion of TGFβ and reduces immunosuppression of T cells and macrophages in the tumor microenvironment. Pan02 tumor-bearing mice were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested (n=4 per group) on day 24 and pooled in pairs among treatment groups. Tumor-conditioned media (TCM) secreted by 0.1×10 6 cells from the tumor digest during a 48 hours culture was harvested. (A) Quantification of TGFβ1 protein levels in TCM from untreated and TGFβ-vaccinated mice. (B) Effect of TCM from untreated or TGFβ-vaccinated mice on the proliferation of anti-CD3/CD28-stimualted naïve splenocytes from an untreated tumor-free mouse. Naïve splenocytes were CFSE-labeled and activated with Dynabeads Mouse T-Activator CD3/CD28 (1:1 ratio) for 48 hours in the presence or absence of TCM generated from tumors from untreated or TGFβ-vaccinated mice. Proliferation of live CD3 + cells was measured by flow cytometry. Gating strategy can be found in . (B, left) Proliferation index in CD3 + cells across culture conditions. (B, right) Representative histograms of CFSE staining in live CD3 + cells (B). (C–E) Effect of TCM from untreated or TGFβ-vaccinated mice on the phenotype of bone-marrow derived macrophages (BMDM) from an untreated tumor-free mouse that were polarized with IL-4 to an M2-like phenotype. BMDM were cultured for 24 hours in the presence of TCM generated from tumors from untreated or TGFβ-vaccinated mice. The phenotype of BMDM was assessed by flow cytometry. (C, top) M1/M2 ratio in BMDM cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. Macrophages were gated as CD11b + F4/80 + , M1 and M2 macrophages were gated as CD11b + F4/80 + mannose receptor (MR) − and CD11b + F4/80 + MR + , respectively. Gating strategy can be found in . (C, bottom) Representative histograms of MR on macrophages across culture conditions shown in (C, top). (D, top) Mean fluorescence intensity (MFI) of Arg1 macrophages cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. (D, bottom) Representative histograms of Arg1 on macrophages across culture conditions shown in (D, top). (E, top) MFI of PD-L1 macrophages cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. (E, bottom) Representative histograms of PD-L1 on macrophages across culture conditions shown in (E, top). Bar plots are presented as mean±SD. Dots represent data derived from TCM generated from pooled tumors (n=2 per group). Arg1, arginase-1; IL, interleukin; PD-L1, programmed death-ligand 1; TGFβ, transforming growth factor-β.

Article Snippet: The following antibodies/dies (purchased from BioLegend unless otherwise stated) were used for flow cytometry: CD45-PE-Cy7, CD31-FITC, FAP-biotin (R&D system), streptavidin-APC, CD90-BV605, PDPN-APC, Ly6C-AF700, CD26-PerCP-Cy5.5 (eBioscience), αSMA-Cy3 (Sigma), CD11b-PE-Cy7, CD11b-Pacific Blue, F4/80-APC, F4/80-FITC, MR-PE, MR-PE-Cy7, Ly6C-PerCP-Cy5.5, Ly6G-APC-Cy7, Arg1-PE (R&D system), PDL1-APC (BD Biosciences), CD45-FITC, CD3-AF700, CD4-BV421, CD8-BV605 (BD Biosciences), CD25-PE-Cy7, FoxP3-APC and carboxyfluorescein succinimidyl ester (CFSE, Sigma Aldrich).

Techniques: Labeling, Generated, Flow Cytometry, Staining, Derivative Assay, Cell Culture, Fluorescence