pe cy7 conjugated antibodies against cd3  (Miltenyi Biotec)

Journal: Arthritis Research & Therapy

Article Title: Proinflammatory T cell polarization is already present in patients with early knee osteoarthritis

doi: 10.1186/s13075-020-02410-w

Figure Lengend Snippet: CD4 + T cell infiltration in the peripheral blood (PB) and joint-derived samples (SF, SM)

Article Snippet: For surface marker expression, PE-Cy7-conjugated mAb against CD3 (clone SK7), APC-Cy7-conjugated mAb against CD4 (clone RPA-T4), and VioBlue-labeled mAb against CD8 (clone BW135/80, Miltenyi Biotec, Germany) were used, and staining was performed at 4 °C for 30 min.

Techniques: Cell Counting

Journal: Arthritis Research & Therapy

Article Title: Proinflammatory T cell polarization is already present in patients with early knee osteoarthritis

doi: 10.1186/s13075-020-02410-w

Figure Lengend Snippet: Flow cytometry analysis of CD4 + T cell surface marker expression. Representative flow cytometry histograms of CD4 + T cell surface marker expression are shown. In short, mononuclear cells were isolated from PB, SF, and SM by density gradient centrifugation. CD3 + MACS-isolated T cells from PB, SF, and SM were stained with monoclonal antibodies (mAb) for the following surface markers: CD4, CD25, CD127, CXCR3, CCR5, CCR3, CCR4, CD161, and CCR6. Before flow cytometric detection, 7-AAD was added to the cell suspensions to exclude cell debris and dead cells. The cutoff for all cell surface markers was defined based on fluorescence minus one (FMO) controls (shown as gray overlay population). mAb, monoclonal antibody; MACS, magnetic-activated cell sorting

Article Snippet: For surface marker expression, PE-Cy7-conjugated mAb against CD3 (clone SK7), APC-Cy7-conjugated mAb against CD4 (clone RPA-T4), and VioBlue-labeled mAb against CD8 (clone BW135/80, Miltenyi Biotec, Germany) were used, and staining was performed at 4 °C for 30 min.

Techniques: Flow Cytometry, Marker, Expressing, Isolation, Gradient Centrifugation, Staining, Bioprocessing, Fluorescence, FACS

Journal: Arthritis Research & Therapy

Article Title: Proinflammatory T cell polarization is already present in patients with early knee osteoarthritis

doi: 10.1186/s13075-020-02410-w

Figure Lengend Snippet: Cytokine secretion of activated CD3 + CD4 + CD8 − T cells in the peripheral blood (PB) and joint-derived samples (SF, SM)

Article Snippet: For surface marker expression, PE-Cy7-conjugated mAb against CD3 (clone SK7), APC-Cy7-conjugated mAb against CD4 (clone RPA-T4), and VioBlue-labeled mAb against CD8 (clone BW135/80, Miltenyi Biotec, Germany) were used, and staining was performed at 4 °C for 30 min.

Techniques:

Journal: Oncoimmunology

Article Title: A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice

doi: 10.1080/2162402X.2017.1421890

Figure Lengend Snippet: PI3K p110α isoform-selective inhibitor combined with anti-neu antibody results in the potentiation of anti-tumor activity, increased T cell infiltration into tumors, and enhanced tumor antigen-specific T cell responses in TUBO tumor model. (A) TUBO cells were treated with 1 μM GDC-0941 or A66 in the absence or presence of anti-neu antibody for 72 h. Cell viability was evaluated with the TOX6 assay kit. Data represent mean ± SEM of triplicate measurements from three independent experiments. ***P < 0.001 vs. vehicle-treated control; ###P < 0.001 vs. PI3K inhibitor alone. (B) BALB/C mice (n = 4–6/group) were s.c. injected with 5 × 105 TUBO cells, and tumor volume was measured. Mice were treated with 200 or 100 μg anti-neu antibody by i.p. injection on days 14 and 17 after tumor cell inoculation, respectively. GDC-0941 (125 mg/kg) or A66 (100 mg/kg) was administered daily by oral gavage from days 14 to 17. (C, D) On day 11 after the first anti-neu antibody treatment (25 days after tumor cell inoculation), tumors and spleens were removed to obtain cell suspensions for surface or intracellular immunolabeling and the ELISPOT assay. (C) Flow cytometry analysis of CD45+, CD4+, and CD8+ cells gated on CD45+ cells and IFN-γ+ cells gated on CD8+ cells in tumors. (D) ELISPOT assay of IFN-γ+ cells using splenocytes co-cultured with BMDCs pulsed with tumor antigen from TUBO cells. Data represent mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, each agent vs. vehicle control; #P < 0.05, ##P < 0.01, ###P < 0.001, each agent vs. anti-neu antibody treatment.

Article Snippet: Antibodies against mouse CD45 (clone 30-F11), CD3 (clone 145-2C11), CD8α (clone 53–6.7), and CD4 (clone GK1.5) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and PE-Cy7, respectively, were purchased from eBioscience (San Diego, CA, USA).

Techniques: Activity Assay, Control, Injection, Immunolabeling, Enzyme-linked Immunospot, Flow Cytometry, Cell Culture

Journal: Oncoimmunology

Article Title: A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice

doi: 10.1080/2162402X.2017.1421890

Figure Lengend Snippet: CD8+ T cells are essential for combination therapy with a PI3K inhibitor and anti-neu antibody in TUBO tumor model. BALB/C mice (n = 3–5/group) were s.c. injected with 5 × 105 TUBO cells, and 200 μg of CD8 or CD4-depleting antibody or control antibody (rat IgG) were administered by i.p. injection on days 17 and 21 after tumor cell inoculation. Mice were treated with 200 or 100 μg anti-neu antibody by i.p. injection on days 18 and 21 after tumor cell injection, respectively, and were orally administered GDC-0941 (125 mg/kg) (A) or A66 (100 mg/kg) (B) daily from days 18 to 21. Tumor volume was measured. Data represent mean ± SEM. *P < 0.05, **P < 0.01 vs. rat IgG group.

Article Snippet: Antibodies against mouse CD45 (clone 30-F11), CD3 (clone 145-2C11), CD8α (clone 53–6.7), and CD4 (clone GK1.5) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and PE-Cy7, respectively, were purchased from eBioscience (San Diego, CA, USA).

Techniques: Injection, Control

Journal: Oncoimmunology

Article Title: A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice

doi: 10.1080/2162402X.2017.1421890

Figure Lengend Snippet: PI3K p110α isoform-selective inhibitor is more effective than pan-PI3K inhibitor in combination with the anti-neu antibody in controlling tumor mass and prolonging survival in a heterogeneous mixed-tumor model. (A) TUBO-P2J cells were treated with various concentrations of anti-neu antibody, GDC-0941, or A66 for 48 h. Cell viability was evaluated with the sulforhodamine B assay. (B, C) To establish the heterogeneous mixed-tumor model, BALB/C mice (n = 5/group) were s.c. inoculated with a mixture of 5 × 105 TUBO cells and 1.25 × 102 TUBO-P2J cells. Mice bearing TUBO and TUBO-P2J mixed tumors were treated with anti-neu antibody and GDC-0941 or A66, as described in the legend of Fig. 1B. (B, C) Tumor volume (B) and survival (C) were monitored. *P < 0.05 vs. vehicle group form day 23 or 25. Data were derived from two independent experiments. (D, E) On day 11 after the first anti-neu antibody treatment, tumors were removed to obtain cell suspensions for surface or intracellular immunolabeling. (D) Flow cytometry analysis of the percentages of CD4+ or CD8+ cells gated on CD45+ cells and IFN-γ+ cells gated on CD8+ cells in tumors. (E) ELISPOT assay of IFN-γ+ cells using splenocytes co-cultured with BMDCs pulsed with tumor antigen from TUBO or TUBO-P2J cells. Data represent mean ± SEM of triplicate determinations. P < 0.05, **P < 0.01, ***P < 0.001, each agent vs. vehicle control; #P < 0.05, ##P < 0.01, ###P < 0.001, each agent vs. anti-neu antibody treatment.

Article Snippet: Antibodies against mouse CD45 (clone 30-F11), CD3 (clone 145-2C11), CD8α (clone 53–6.7), and CD4 (clone GK1.5) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and PE-Cy7, respectively, were purchased from eBioscience (San Diego, CA, USA).

Techniques: Sulforhodamine B Assay, Derivative Assay, Immunolabeling, Flow Cytometry, Enzyme-linked Immunospot, Cell Culture, Control

Journal: Oncoimmunology

Article Title: A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice

doi: 10.1080/2162402X.2017.1421890

Figure Lengend Snippet: CD8+ T cells are indispensable for combination therapy with a PI3K inhibitor and anti-neu antibody in the HER2/neu IHC2+ tumor model. (A, B) BALB/C mice (n = 3–5/group) were s.c. injected with a mixture of 5 × 105 TUBO and 1.25 × 102 TUBO-P2J cells; 200 μg CD8 or CD4-depleting antibody or control antibody (rat IgG) were administered by i.p. injection on days 14 and 18 after tumor cell inoculation. Mice were treated with 200 or 100 μg anti-neu antibody by i.p. injection on days 15 and 18 after tumor cell inoculation, respectively, and were orally administered GDC-0941 (125 mg/kg) (A) or A66 (100 mg/kg) (B) daily from days 15 to 18. Tumor volume was measured. Data represent mean ± SEM. *P < 0.05, **P < 0.01 vs. rat IgG group.

Article Snippet: Antibodies against mouse CD45 (clone 30-F11), CD3 (clone 145-2C11), CD8α (clone 53–6.7), and CD4 (clone GK1.5) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and PE-Cy7, respectively, were purchased from eBioscience (San Diego, CA, USA).

Techniques: Injection, Control

Journal: Oncoimmunology

Article Title: A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice

doi: 10.1080/2162402X.2017.1421890

Figure Lengend Snippet: PI3K p110α isoform-selective inhibitor preserves the anti-neu antibody-induced increase in pAKT+ T cell numbers in TUBO and heterogeneous mixed tumor models. (A) Mice bearing TUBO tumors were treated with anti-neu antibody alone or in combination with PI3K inhibitor. On day 11 after the first anti-neu antibody treatment (25 days after tumor cell inoculation), tumors were removed to obtain cell suspensions for intracellular immunolabelling as described in the legend for Fig. 1C. (B) Mice bearing TUBO and TUBO-P2J mixed-tumors were treated with anti-neu antibody and GDC-0941 or A66. On day 11 after the first anti-neu antibody treatment, tumors were removed to obtain cell suspensions for intracellular immunolabelling as described in the legend for Fig. 3D. The number of pAKT+ cells in tumors was determined by flow cytometry. Intracellular labeling of pAKT (S473) in the CD8+ T cell subset of tumors was analyzed. Data are represent mean ± SEM. P < 0.05, **P < 0.01, ***P < 0.001, each agent vs. vehicle control; #P < 0.05, ##P < 0.01, ###P < 0.001, each agent vs. anti-neu antibody treatment.

Article Snippet: Antibodies against mouse CD45 (clone 30-F11), CD3 (clone 145-2C11), CD8α (clone 53–6.7), and CD4 (clone GK1.5) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and PE-Cy7, respectively, were purchased from eBioscience (San Diego, CA, USA).

Techniques: Flow Cytometry, Labeling, Control

Journal: Oncoimmunology

Article Title: A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice

doi: 10.1080/2162402X.2017.1421890

Figure Lengend Snippet: PI3K p110α isoform-selective inhibitor preserves antigen-specific or non-specific T cell proliferation. (A) Splenocytes isolated from mice inoculated with HA-specific CD8+ (CL4) T cells were labeled with CFSE and then stimulated with 0.1 μg/ml HA512-520 peptide in the presence of A66 or GDC-0941 for 48 or 72 h. (B) Splenocytes isolated from naïve BALB/C mice were labeled with CFSE and then stimulated with the 0.5 μg/ml CD3/CD28 antibody in the presence of A66 or GDC-0941 for 48 or 72 h. HA antigen-specific or non-specific T cell proliferation was evaluated based on CFSE dilution by flow cytometry. Data represent mean ± SEM of three independent experiments. ***P < 0.001.

Article Snippet: Antibodies against mouse CD45 (clone 30-F11), CD3 (clone 145-2C11), CD8α (clone 53–6.7), and CD4 (clone GK1.5) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and PE-Cy7, respectively, were purchased from eBioscience (San Diego, CA, USA).

Techniques: Isolation, Labeling, Flow Cytometry