CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), PE-Cyanine7, eBioscience™

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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.

Protocol tips

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.

Publication protocol

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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