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Found 3 matching solutions for this experiment
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7AAD (Biolegend) or Zombie Aqua Fixable Viability Kit (Biolegend) was used to exclude dead cells. For intracellular transcription factor staining, surface-stained cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer's protocol |
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| Protocol tips |
| 7AAD (Biolegend) or Zombie Aqua Fixable Viability Kit (Biolegend) was used to exclude dead cells. For intracellular transcription factor staining, surface-stained cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer's protocol |
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To obtain single-cell suspensions, tumor tissues were digested by 1 mg/ml collagenase IV (Sigma-Aldrich) and 0.2 mg/ml DNase I (Sigma-Aldrich) for 45 minutes at 37°C. Cells were blocked with anti-FcR (clone 2.4G2; Bio-Xcell) and then stained with antibodies against PD-L1, PD-1, CD11b, Gr1, F4/80, CD11c, CD8, CD4, Foxp3, and CD45 (BioLegend). For apoptosis assays, MDSCs were harvested, blockaded with anti-FcR, and stained with antibodies against Ly6C, CD11b, and annexin V. Samples were collected on a FACSCalibur Flow Cytometer (BD), and data were analyzed using FlowJo software (Tree Star Inc.). |
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| To obtain single-cell suspensions, tumor tissues were digested by 1 mg/ml collagenase IV (Sigma-Aldrich) and 0.2 mg/ml DNase I (Sigma-Aldrich) for 45 minutes at 37°C. Cells were blocked with anti-FcR (clone 2.4G2; Bio-Xcell) and then stained with antibodies against PD-L1, PD-1, CD11b, Gr1, F4/80, CD11c, CD8, CD4, Foxp3, and CD45 (BioLegend). For apoptosis assays, MDSCs were harvested, blockaded with anti-FcR, and stained with antibodies against Ly6C, CD11b, and annexin V. Samples were collected on a FACSCalibur Flow Cytometer (BD), and data were analyzed using FlowJo software (Tree Star Inc.). |
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For fluorescence-activated cell sorting (FACS) analysis of brain tumor-infiltrating immune cells, mice were euthanized at the defined endpoint. Mononuclear cells in the brains were isolated as previously described and stained afterward with the respective antibodies for FACS analysis. For flow cytometry, cells were counted and incubated with Fc blocker (eBiosciences, USA) for 30 min, followed by another 30-min incubation with conjugated antibodies for extracellular markers. For intracellular cytokine detection, cells were stimulated in vitro with Cell Stimulation Cocktail (eBiosciences, USA) for 5 h at 37 °C before FACS analysis. After stimulation, cells were stained for surface markers and cytokines with Intracellular Fixation and Permeabilization Buffer Set (eBiosciences, USA). All antibodies used for these experiments were listed in Additional file 1: Table S2. Proper isotype controls and compensation controls were performed in parallel. BD Biosciences Canto II (BD Biosciences, USA) was used for flow cytometry. FlowJo software (Tree Star, USA) was used for FACS data analysis. |
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| For fluorescence-activated cell sorting (FACS) analysis of brain tumor-infiltrating immune cells, mice were euthanized at the defined endpoint. Mononuclear cells in the brains were isolated as previously described and stained afterward with the respective antibodies for FACS analysis. For flow cytometry, cells were counted and incubated with Fc blocker (eBiosciences, USA) for 30 min, followed by another 30-min incubation with conjugated antibodies for extracellular markers. For intracellular cytokine detection, cells were stimulated in vitro with Cell Stimulation Cocktail (eBiosciences, USA) for 5 h at 37 °C before FACS analysis. After stimulation, cells were stained for surface markers and cytokines with Intracellular Fixation and Permeabilization Buffer Set (eBiosciences, USA). All antibodies used for these experiments were listed in Additional file 1: Table S2. Proper isotype controls and compensation controls were performed in parallel. BD Biosciences Canto II (BD Biosciences, USA) was used for flow cytometry. FlowJo software (Tree Star, USA) was used for FACS data analysis. |
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