Cell Isolation:Article Title: Tumor antigens preferentially derive from unmutated genomic sequences in melanoma and non-small cell lung cancer
Article Snippet: CD8 + T cells from D50 (leukapheresis product from a healthy HLA-A*02:01-positive donor from BioIVT) were expanded in eight different conditions: with individual aeTSAs (VLMMKLEDL, RLLELHITM, VLWRGDSPL, YQIGQVQGV), control peptides (MelanA26-35A27L, ELAGIGILTV; pp65495–503, NLVPMVATV), an aeTSA from ovarian cancer (Ov-aeTSA, LLSSKLLLM) described as immunogenic by our group , all presented by HLA-A*02:01, or DMSO as the negative control. .. Thawed PBMCs were enriched for naive CD8 + T cells using the Human CD8 + T Cell Isolation Kit combined with CD45RO and CD57 MicroBeads (Miltenyi Biotec) to remove memory T cells from the CD8 + T cell fraction. .. These naive CD8 + T cells were then expanded for 14 d with T Cell TransAct (Miltenyi Biotec) in TexMACS Medium (Miltenyi Biotec) supplemented with interleukin (IL)-7 and IL-21 (PeproTech, at final concentrations of 5 ng ml −1 and 10 ng ml −1 , respectively) before specific expansion.
Article Title: CDK4/6 inhibition reprograms the breast cancer immunopeptidome via Rb-dependent chromatin and transcriptomic remodeling.
Article Snippet: .. Thawed PBMCs were enriched for naive CD8 + T cells using the Human CD8 + T cell Isolation Kit combined with CD45RO and CD57 MicroBeads (Miltenyi Biotec) to deplete memory T cells. .. These naive CD8 + T cells were then expanded for 14 days with T cell TransAct (Miltenyi Biotec) in TexMACS Medium (Miltenyi Biotec) supplemented with interleukin-7 (5 ng/mL, Thermo Fisher Scientific) and interleukin-21 (10 ng/mL, PeproTech).
Article Title: Tumor antigens preferentially derive from unmutated genomic sequences in melanoma and non-small cell lung cancer.
Article Snippet: CD8+ T cells from D50 (leukapheresis product from a healthy HLA-A*02:01-positive donor from BioIVT) were expanded in eight different conditions: with individual aeTSAs (VLMMKLEDL, RLLELHITM, VLWRGDSPL, YQIGQVQGV), control peptides (MelanA26-35A27L, ELAGIGILTV; pp65495–503, NLVPMVATV), an aeTSA from ovarian cancer (Ov-aeTSA, LLSSKLLLM) described as immunogenic by our group92, all presented by HLA-A*02:01, or DMSO as the negative control. .. Thawed PBMCs were enriched for naive CD8+ T cells using the Human CD8+ T Cell Isolation Kit combined with CD45RO and CD57 MicroBeads (Miltenyi Biotec) to remove memory T cells from the CD8+ T cell fraction. .. These naive CD8+ T cells were then expanded for 14 d with T Cell TransAct (Miltenyi Biotec) in TexMACS Medium (Miltenyi Biotec) supplemented with interleukin (IL)-7 and IL-21 (PeproTech, at final concentrations of 5 ng ml−1 and 10 ng ml−1, respectively) before specific expansion.
Imaging:Article Title: Inhibition of autophagy enhances the antitumor efficacy of T/CAR T cell against neuroblastoma
Article Snippet: Fluorochromes were photobleached or released using REAlease technology (Miltenyi) after image acquisition at the end of each staining cycle. .. The following antibodies (all from Miltenyi Biotec) were used: Ki67, CD3, CD4, CD8, CD44, CD45RA, CD45RO, CD57, CD183, CD196, LAG3 (CD223), PD-1 (CD279), TIM3 (CD366), FoxP3, HLA-DR. Acquired images were processed and analyzed by MACSiQ ® View Imaging Software (Miltenyi Biotec) following current processing work-flow (Miltenyi Biotec). ..
Software:Article Title: Inhibition of autophagy enhances the antitumor efficacy of T/CAR T cell against neuroblastoma
Article Snippet: Fluorochromes were photobleached or released using REAlease technology (Miltenyi) after image acquisition at the end of each staining cycle. .. The following antibodies (all from Miltenyi Biotec) were used: Ki67, CD3, CD4, CD8, CD44, CD45RA, CD45RO, CD57, CD183, CD196, LAG3 (CD223), PD-1 (CD279), TIM3 (CD366), FoxP3, HLA-DR. Acquired images were processed and analyzed by MACSiQ ® View Imaging Software (Miltenyi Biotec) following current processing work-flow (Miltenyi Biotec). ..
Staining:Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
Expressing:Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
Construct:Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
Incubation:Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
Labeling:Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
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